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1
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Zhang T, Cai R, Sun C. Light and polyphosphate kinase 2 cooperatively regulate the production of zero-valent sulfur in a deep-sea bacterium. mSystems 2025; 10:e0047325. [PMID: 40377319 DOI: 10.1128/msystems.00473-25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2025] [Accepted: 04/21/2025] [Indexed: 05/18/2025] Open
Abstract
It is well established that different wavelengths of light exist in various deep-sea environments, and many deep-sea microorganisms have evolved specialized mechanisms for sensing and utilizing light energy. Our previous research found that blue light promotes zero-valent sulfur (ZVS) production in Erythrobacter flavus 21-3, a bacterium isolated from a deep-sea cold seep. Given that long-wavelength light is more prevalent in deep-sea environments, the present study investigates the mechanism by which E. flavus 21-3 senses infrared light (wavelength 940 nm) and regulates ZVS production. We found that the bacteriophytochrome BPHP-15570 is responsible for sensing infrared light, which induces autophosphorylation of BPHP-15570, activating the diguanylate cyclase DGC-0450 for c-di-GMP biosynthesis. Subsequently, the PilZ domain-containing protein mPilZ-1753 binds to c-di-GMP, triggering a well-established ZVS production pathway involving thiosulfate dehydrogenase (TsdA) and two homologs of thiosulfohydrolases (SoxB). Notably, polyphosphate kinase 2 (PPK2) is recruited to compete for GTP, the direct precursor of c-di-GMP biosynthesis. This competition downregulates ZVS production as well as other important metabolic processes. This negative regulatory pathway helps the bacterium avoid excessive ZVS accumulation, which could be toxic to bacterial growth. Overall, E. flavus 21-3 has evolved a sophisticated regulatory pathway to sense both blue and infrared light, triggering ZVS production. Our study provides a valuable model for understanding light utilization and its coupling with sulfur cycling in deep-sea environments.IMPORTANCEIt is widely believed that deep-sea ecosystems operate independently of light, relying primarily on chemical energy. However, the discovery of non-photosynthetic bacteria in various deep-sea environments that can sense and utilize light has challenged this assumption. In a recent study, we found that blue light significantly promotes the production of zero-valent sulfur (ZVS) in the deep-sea bacterium Erythrobacter flavus 21-3. Given that long-wavelength light is more prevalent in deep-sea environments, we investigated whether infrared light also plays a role in regulating ZVS production in E. flavus 21-3. Our results indicate that infrared light does promote ZVS formation in this bacterium. We identified PPK2 as a negative regulator, maintaining intracellular ZVS at safe levels to prevent toxicity due to excessive accumulation. Overall, our study offers a valuable model for exploring how light is utilized and its interaction with microbial sulfur cycling in the extreme conditions of the deep sea.
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Affiliation(s)
- Tianhang Zhang
- CAS and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China
- College of Earth Science, University of Chinese Academy of Sciences, Beijing, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China
| | - Ruining Cai
- CAS and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China
| | - Chaomin Sun
- CAS and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China
- College of Earth Science, University of Chinese Academy of Sciences, Beijing, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China
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2
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Lakshmi MA, Kulshreshtha A, Mondal KK, Dasgupta I, Tyagi A, Kumar S, Kalaivanan NS, Mrutyunjaya S, Sreenayana B, Rashmi ER, Ghoshal T, Jagram N, Challa GK, Mani C. Functional validation of OsRPM1 as a positive regulator of bacterial blight resistance in rice via virus-induced gene silencing. Folia Microbiol (Praha) 2025:10.1007/s12223-025-01280-6. [PMID: 40490614 DOI: 10.1007/s12223-025-01280-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Accepted: 05/14/2025] [Indexed: 06/11/2025]
Abstract
Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a major constraint to rice production in humid tropical regions. In the search for new genetic sources of resistance, we focused on OsRPM1 (LOC Os12g30070.1), a rice gene encoding a coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) protein, structurally similar to well-characterized resistance (R) proteins in Arabidopsis and other plant species. Although its role in rice immunity was previously uncharacterized, transcriptomic profiling revealed that OsRPM1 is significantly upregulated in a type III secretion system (T3SS)-dependent manner during infection with the virulent Xoo race 4, suggesting a pathogen-responsive defence function. To evaluate this, we employed virus-induced gene silencing (VIGS) to transiently suppress its expression in rice. Silencing OsRPM1 increased susceptibility to Xoo, resulting in more severe disease symptoms, reduced reactive oxygen species (ROS) accumulation, and impaired callose deposition; key defence responses linked to effective resistance. These findings demonstrate that OsRPM1 acts as a positive regulator of rice defence and support its potential as a candidate for broad-spectrum, durable resistance breeding.
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Affiliation(s)
- M Amrutha Lakshmi
- ICAR-Indian Institute of Oil Palm Research, Eluru, Andhra Pradesh, 534435, India
| | | | - Kalyan K Mondal
- ICAR-National Institute of Biotic Stress Management, Raipur, Chhattisgarh, 493225, India.
| | - Indranil Dasgupta
- Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, India
| | - Aditya Tyagi
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - Sanjeev Kumar
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - N S Kalaivanan
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - S Mrutyunjaya
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - B Sreenayana
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - E R Rashmi
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - Thungri Ghoshal
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
| | - Neelam Jagram
- Department of Plant Molecular Biology, University of Delhi, South Campus, New Delhi, 110021, India
| | - G K Challa
- ICAR-Indian Institute of Oil Palm Research, Eluru, Andhra Pradesh, 534435, India
| | - Chandra Mani
- Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India
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3
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Müller GA. The Transformation Experiment of Frederick Griffith II: Inclusion of Cellular Heredity for the Creation of Novel Microorganisms. Bioengineering (Basel) 2025; 12:532. [PMID: 40428151 PMCID: PMC12109375 DOI: 10.3390/bioengineering12050532] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2025] [Revised: 05/05/2025] [Accepted: 05/07/2025] [Indexed: 05/29/2025] Open
Abstract
So far, synthetic biology approaches for the construction of artificial microorganisms have fostered the transformation of acceptor cells with genomes from donor cells. However, this strategy seems to be limited to closely related bacterial species only, due to the need for a "fit" between donor and acceptor proteomes and structures. "Fitting" of cellular regulation of metabolite fluxes and turnover between donor and acceptor cells, i.e. cybernetic heredity, may be even more difficult to achieve. The bacterial transformation experiment design 1.0, as introduced by Frederick Griffith almost one century ago, may support integration of DNA, macromolecular, topological, cybernetic and cellular heredity: (i) attenuation of donor Pneumococci of (S) serotype fosters release of DNA, and hypothetically of non-DNA structures compatible with subsequent transfer to and transformation of acceptor Pneumococci from (R) to (S) serotype; (ii) use of intact donor cells rather than of subcellular or purified fractions may guarantee maximal diversity of the structural and cybernetic matter and information transferred; (iii) "Blending" or mixing and fusion of donor and acceptor Pneumococci may occur under accompanying transfer of metabolites and regulatory circuits. A Griffith transformation experiment design 2.0 is suggested, which may enable efficient exchange of DNA as well as non-DNA structural and cybernetic matter and information, leading to unicellular hybrid microorganisms with large morphological/metabolic phenotypic differences and major features compared to predeceding cells. The prerequisites of horizontal gene and somatic cell nuclear transfer, the molecular mechanism of transformation, the machineries for the biogenesis of bacterial cytoskeleton, micelle-like complexes and membrane landscapes are briefly reviewed on the basis of underlying conceptions, ranging from Darwin's "gemmules" to "stirps", cytoplasmic and "plasmon" inheritance, "rhizene agency", "communicology", "transdisciplinary membranology" to up to Kirschner's "facilitated variation".
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Affiliation(s)
- Günter A. Müller
- Biology and Technology Studies Institute Munich (BITSIM), 80939 Munich, Germany; ; Tel.: +49-151-25216987
- Institute of Media Sociology, Department of Cultural Sciences, University of Paderborn, 33104 Paderborn, Germany
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4
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Yamagishi K, Ike M, Tokuyasu K. Construction of a genome-editing system for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain. Biosci Biotechnol Biochem 2024; 89:80-87. [PMID: 39533823 DOI: 10.1093/bbb/zbae157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Accepted: 10/23/2024] [Indexed: 11/16/2024]
Abstract
Thermophilic actinomycetes significantly contribute to the terrestrial carbon cycle via the rapid degradation of lignocellulosic polysaccharides in composts. In this study, a genome-editing system was constructed for the thermophilic actinomycete Streptomyces thermodiastaticus K5 strain, which was isolated from compost. The genome-editing plasmid (pGEK5) harboring nickase Cas9 was derived from the high-copy plasmid pL99 and used for the K5 strain. It was found that pGEK5 could easily be lost from the transformed clone through cultivation on apramycin-free medium and spore formation, enabling its reuse for subsequent genome-editing cycles. With the aid of this plasmid, mutations were sequentially introduced to 2 uracil-DNA glycosylase genes (Udg1 and Udg2) and 1 β-glucosidase gene (Bgl1). Thus, the genome-editing system using pGEK5 enables us to start the functional modification of this thermophilic actinomycete, especially for improved conversion of lignocellulosic biomass.
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Affiliation(s)
- Kenji Yamagishi
- Institute of Food Research, National Agriculture and Food Research Organization, Japan
| | - Masakazu Ike
- Institute of Food Research, National Agriculture and Food Research Organization, Japan
| | - Ken Tokuyasu
- Institute of Food Research, National Agriculture and Food Research Organization, Japan
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5
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Tanaka R, Tamao K, Ono M, Yamayoshi S, Kawaoka Y, Su'etsugu M, Noji H, Tabata KV. In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system. PLoS One 2024; 19:e0312776. [PMID: 39514509 PMCID: PMC11548778 DOI: 10.1371/journal.pone.0312776] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2024] [Accepted: 10/13/2024] [Indexed: 11/16/2024] Open
Abstract
The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E.coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method enables the pathogenicity analysis and vaccine development using genetically modified viruses, and it is expected to allow for faster analysis of newly emerging variants than ever before. Furthermore, its application to other RNA viruses is also expected.
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Affiliation(s)
- Ryota Tanaka
- Department of Applied Chemistry, Graduate School of Engineering, the University of Tokyo, Tokyo, Japan
| | - Kenji Tamao
- Department of Applied Chemistry, Graduate School of Engineering, the University of Tokyo, Tokyo, Japan
| | - Mana Ono
- Department of Applied Chemistry, Graduate School of Engineering, the University of Tokyo, Tokyo, Japan
| | - Seiya Yamayoshi
- Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan
| | - Yoshihiro Kawaoka
- Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan
- The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan
- Department of Pathobiological Sciences, Influenza Research Institute, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin, United States of America
- Pandemic Preparedness, Infection and Advanced Research Center (UTOPIA), University of Tokyo, Tokyo, Japan
| | - Masayuki Su'etsugu
- Department of Life Science, College of Science, Rikkyo University, Tokyo, Japan
| | - Hiroyuki Noji
- Department of Applied Chemistry, Graduate School of Engineering, the University of Tokyo, Tokyo, Japan
| | - Kazuhito V Tabata
- Department of Applied Chemistry, Graduate School of Engineering, the University of Tokyo, Tokyo, Japan
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6
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Abe K, Yahara H, Nakao R, Yamaguchi T, Akeda Y. A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence. Front Microbiol 2024; 15:1476171. [PMID: 39498132 PMCID: PMC11532111 DOI: 10.3389/fmicb.2024.1476171] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2024] [Accepted: 10/03/2024] [Indexed: 11/07/2024] Open
Abstract
Porphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gingivalis; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for P. gingivalis without any special equipment by exploiting its natural DNA competence. P. gingivalis ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 106 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other P. gingivalis strains, W83 and TDC60. Additionally, we discovered that deletion of PGN_0421 or PGN_0519, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.
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Affiliation(s)
- Kimihiro Abe
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hiroko Yahara
- Genome Medical Science Project, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan
| | - Ryoma Nakao
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takehiro Yamaguchi
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yukihiro Akeda
- Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan
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7
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Wang Z, Zeng Y, Ahmed Z, Qin H, Bhatti IA, Cao H. Calcium‐dependent antimicrobials: Nature‐inspired materials and designs. EXPLORATION (BEIJING, CHINA) 2024; 4:20230099. [PMID: 39439493 PMCID: PMC11491315 DOI: 10.1002/exp.20230099] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Accepted: 02/02/2024] [Indexed: 10/25/2024]
Abstract
Bacterial infection remains a major complication answering for the failures of various implantable medical devices. Tremendous extraordinary advances have been published in the design and synthesis of antimicrobial materials addressing this issue; however, the clinical translation has largely been blocked due to the challenge of balancing the efficacy and safety of these materials. Here, calcium's biochemical features, natural roles in pathogens and the immune systems, and advanced uses in infection medications are illuminated, showing calcium is a promising target for developing implantable devices with less infection tendency. The paper gives a historical overview of biomedical uses of calcium and summarizes calcium's merits in coordination, hydration, ionization, and stereochemistry for acting as a structural former or trigger in biological systems. It focuses on the involvement of calcium in pathogens' integrity, motility, and metabolism maintenance, outlining the potential antimicrobial targets for calcium. It addresses calcium's uses in the immune systems that the authors can learn from for antimicrobial synthesis. Additionally, the advances in calcium's uses in infection medications are highlighted to sketch the future directions for developing implantable antimicrobial materials. In conclusion, calcium is at the nexus of antimicrobial defense, and future works on taking advantage of calcium in antimicrobial developments are promising in clinical translation.
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Affiliation(s)
- Zhong Wang
- Interfacial Electrochemistry and BiomaterialsSchool of Materials Science and EngineeringEast China University of Science and TechnologyShanghaiChina
| | - Yongjie Zeng
- Interfacial Electrochemistry and BiomaterialsSchool of Materials Science and EngineeringEast China University of Science and TechnologyShanghaiChina
| | - Zubair Ahmed
- Interfacial Electrochemistry and BiomaterialsSchool of Materials Science and EngineeringEast China University of Science and TechnologyShanghaiChina
| | - Hui Qin
- Department of OrthopaedicsShanghai Jiaotong University Affiliated Sixth People's HospitalShanghaiChina
| | | | - Huiliang Cao
- Interfacial Electrochemistry and BiomaterialsSchool of Materials Science and EngineeringEast China University of Science and TechnologyShanghaiChina
- Engineering Research Center for Biomedical Materials of Ministry of EducationEast China University of Science and TechnologyShanghaiChina
- Key Laboratory for Ultrafine Materials of Ministry of EducationEast China University of Science & TechnologyShanghaiChina
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8
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Ekiz DO, Comlekcioglu U, Comlekcioglu N, Aygan A. Cloning, characterization and functional analysis of lichenase produced by Bacillus licheniformis RB16 isolated from cattle faeces. AN ACAD BRAS CIENC 2024; 96:e20231156. [PMID: 39319834 DOI: 10.1590/0001-3765202420231156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 04/26/2024] [Indexed: 09/26/2024] Open
Abstract
Lichenan, 1,3-1,4-β-Glucan, a linear polysaccharide exists in the cell walls of various cereals, has garnered attention for its industrial applications due to its enzymatic breakdown by lichenase enzymes. In this study, Bacillus licheniformis strain RB16, isolated from cattle faeces, was identified as a robust lichenase producer. The lichenase gene, licA, was successfully cloned and characterized. The cloned RB16 lichenase (LicA) demonstrated its highest activity level at pH 7.5. It also retained over 50% of its activity within the pH range of 6.0-8.5 but experienced a decline to 40% at pH 9.0. LicA was active at temperatures ranging from 25 to 65 °C with an optimum at 45 °C. LicA exhibited more than 60% of its activity at the temperature range of 35-55 °C. Zymogram analysis confirmed LicA's lichenan-degrading ability and structural analysis revealed a stable enzyme structure primarily composed of random coils and extended strands. Although LicA exhibited low thermostability, consistent with its relatively low α-helix content, it demonstrated promising industrial potential. Evolutionary analysis placed LicA within a cluster of closely related Bacillus lichenases, particularly B. halotolerans, B. atrophaeus, and B. spizizenii. These findings expand our understanding of lichenases of Bacillus and underscore its potential for various industrial applications.
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Affiliation(s)
- Dilek Ozgun Ekiz
- Kahramanmaras Sutcu Imam University, Science Faculty, Biology Department, 46040 Kahramanmaras, Turkiye
| | - Ugur Comlekcioglu
- Osmaniye Korkut Ata University, Engineering and Natural Sciences Faculty, Biology Department, 80000 Osmaniye, Turkiye
| | - Nazan Comlekcioglu
- Kahramanmaras Sutcu Imam University, Science Faculty, Biology Department, 46040 Kahramanmaras, Turkiye
| | - Ashabil Aygan
- Kahramanmaras Sutcu Imam University, Science Faculty, Biology Department, 46040 Kahramanmaras, Turkiye
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9
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Chen X, Zhu N, Yang G, Guo X, Sun S, Leng F, Wang Y. Role of cspA on the Preparation of Escherichia coli Competent Cells by Calcium Chloride Method. J Basic Microbiol 2024; 64:e2400113. [PMID: 38924123 DOI: 10.1002/jobm.202400113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2024] [Revised: 06/01/2024] [Accepted: 06/04/2024] [Indexed: 06/28/2024]
Abstract
One of the fundamental techniques in genetic engineering is the creation of Escherichia coli competent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5α competent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA-deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA-deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA-deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET-32a, and p1304, with enhancements of 2.23, 2.24, and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells.
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Affiliation(s)
- Xiaona Chen
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
| | - Ning Zhu
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
- School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China
| | - Guangrui Yang
- Gansu Zhongshang Food Quality Test and Detection Co. Ltd., Lanzhou, China
- Gansu Business Science and Technology Institute Co. Ltd., Lanzhou, China
| | - Xiaopeng Guo
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
| | - Shangchen Sun
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
- School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China
| | - Feifan Leng
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
| | - Yonggang Wang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, China
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10
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Rosch T, Tenhaef J, Stoltmann T, Redeker T, Kösters D, Hollmann N, Krumbach K, Wiechert W, Bott M, Matamouros S, Marienhagen J, Noack S. AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering. ACS Synth Biol 2024; 13:2227-2237. [PMID: 38975718 PMCID: PMC11264319 DOI: 10.1021/acssynbio.4c00298] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Revised: 07/02/2024] [Accepted: 07/02/2024] [Indexed: 07/09/2024]
Abstract
The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.
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Affiliation(s)
- Tobias
Michael Rosch
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Julia Tenhaef
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Tim Stoltmann
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Till Redeker
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Dominic Kösters
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
- Institute
of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany
| | - Niels Hollmann
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
- Institute
of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany
| | - Karin Krumbach
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Wolfgang Wiechert
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Michael Bott
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
- The
Bioeconomy Science Center (BioSC), Forschungszentrum
Jülich, D-52425 Jülich, Germany
| | - Susana Matamouros
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
| | - Jan Marienhagen
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
- Institute
of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany
| | - Stephan Noack
- Institute
of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany
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11
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Maleckis M, Wibowo M, Williams SE, Gotfredsen CH, Sigrist R, Souza LDO, Cowled MS, Charusanti P, Gren T, Saha S, Moreira JMA, Weber T, Ding L. Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus. ACS Chem Biol 2024; 19:1303-1310. [PMID: 38743035 DOI: 10.1021/acschembio.4c00121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.
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Affiliation(s)
- Matiss Maleckis
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Mario Wibowo
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs. Lyngby, Denmark
| | - Sam E Williams
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Charlotte H Gotfredsen
- Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, 2800 Kgs. Lyngby, Denmark
| | - Renata Sigrist
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Luciano D O Souza
- Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
- Sino-Danish Center for Education and Research (SDC), Aarhus University, Dalgas Avenue 4, Building 3410, 8000 Aarhus C, Denmark
| | - Michael S Cowled
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs. Lyngby, Denmark
| | - Pep Charusanti
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Tetiana Gren
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Subhasish Saha
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - José M A Moreira
- Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
| | - Tilmann Weber
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Søltofts Plads, Building 220, 2800 Kgs. Lyngby, Denmark
| | - Ling Ding
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 221, 2800 Kgs. Lyngby, Denmark
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12
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Tenjo-Castaño F, Sofos N, Stutzke LS, Temperini P, Fuglsang A, Pape T, Mesa P, Montoya G. Conformational landscape of the type V-K CRISPR-associated transposon integration assembly. Mol Cell 2024; 84:2353-2367.e5. [PMID: 38834066 DOI: 10.1016/j.molcel.2024.05.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2023] [Revised: 03/11/2024] [Accepted: 05/07/2024] [Indexed: 06/06/2024]
Abstract
CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
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Affiliation(s)
- Francisco Tenjo-Castaño
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Nicholas Sofos
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Luisa S Stutzke
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Piero Temperini
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Anders Fuglsang
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Tillmann Pape
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences University of Copenhagen; Blegdamsvej 3B, DK-2200 Copenhagen, Denmark
| | - Pablo Mesa
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark
| | - Guillermo Montoya
- Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
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13
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Barroso GT, Garcia AA, Knapp M, Boggs DG, Bridwell-Rabb J. Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins. Methods Enzymol 2024; 703:215-242. [PMID: 39260997 DOI: 10.1016/bs.mie.2024.05.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/13/2024]
Abstract
The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.
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Affiliation(s)
- Gage T Barroso
- Department of Chemistry, University of Michigan, Ann Arbor, MI, United States
| | | | - Madison Knapp
- Department of Chemistry, University of Michigan, Ann Arbor, MI, United States
| | - David G Boggs
- Department of Chemistry, University of Michigan, Ann Arbor, MI, United States
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14
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Hu Y, Liu G, Sun C, Wu S. Volatile Organic Compounds Produced by a Deep-Sea Bacterium Efficiently Inhibit the Growth of Pseudomonas aeruginosa PAO1. Mar Drugs 2024; 22:233. [PMID: 38786624 PMCID: PMC11122958 DOI: 10.3390/md22050233] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2024] [Revised: 05/16/2024] [Accepted: 05/17/2024] [Indexed: 05/25/2024] Open
Abstract
The deep-sea bacterium Spongiibacter nanhainus CSC3.9 has significant inhibitory effects on agricultural pathogenic fungi and human pathogenic bacteria, especially Pseudomonas aeruginosa, the notorious multidrug-resistant pathogen affecting human public health. We demonstrate that the corresponding antibacterial agents against P. aeruginosa PAO1 are volatile organic compounds (VOCs, namely VOC-3.9). Our findings show that VOC-3.9 leads to the abnormal cell division of P. aeruginosa PAO1 by disordering the expression of several essential division proteins associated with septal peptidoglycan synthesis. VOC-3.9 hinders the biofilm formation process and promotes the biofilm dispersion process of P. aeruginosa PAO1 by affecting its quorum sensing systems. VOC-3.9 also weakens the iron uptake capability of P. aeruginosa PAO1, leading to reduced enzymatic activity associated with key metabolic processes, such as reactive oxygen species (ROS) scavenging. Overall, our study paves the way to developing antimicrobial compounds against drug-resistant bacteria by using volatile organic compounds.
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Affiliation(s)
- Yuanyuan Hu
- College of Life Sciences, Qingdao University, 308 Ningxia Road, Qingdao 266071, China;
- CAS Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266071, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
| | - Ge Liu
- CAS Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266071, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
| | - Chaomin Sun
- CAS Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266071, China
- Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
- College of Earth Science, University of Chinese Academy of Sciences, Beijing 101408, China
| | - Shimei Wu
- College of Life Sciences, Qingdao University, 308 Ningxia Road, Qingdao 266071, China;
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15
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Reiter MA, Bradley T, Büchel LA, Keller P, Hegedis E, Gassler T, Vorholt JA. A synthetic methylotrophic Escherichia coli as a chassis for bioproduction from methanol. Nat Catal 2024; 7:560-573. [PMID: 38828428 PMCID: PMC11136667 DOI: 10.1038/s41929-024-01137-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2023] [Accepted: 02/29/2024] [Indexed: 06/05/2024]
Abstract
Methanol synthesized from captured greenhouse gases is an emerging renewable feedstock with great potential for bioproduction. Recent research has raised the prospect of methanol bioconversion to value-added products using synthetic methylotrophic Escherichia coli, as its metabolism can be rewired to enable growth solely on the reduced one-carbon compound. Here we describe the generation of an E. coli strain that grows on methanol at a doubling time of 4.3 h-comparable to many natural methylotrophs. To establish bioproduction from methanol using this synthetic chassis, we demonstrate biosynthesis from four metabolic nodes from which numerous bioproducts can be derived: lactic acid from pyruvate, polyhydroxybutyrate from acetyl coenzyme A, itaconic acid from the tricarboxylic acid cycle and p-aminobenzoic acid from the chorismate pathway. In a step towards carbon-negative chemicals and valorizing greenhouse gases, our work brings synthetic methylotrophy in E. coli within reach of industrial applications.
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Affiliation(s)
- Michael A. Reiter
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Timothy Bradley
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Lars A. Büchel
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Philipp Keller
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Emese Hegedis
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Thomas Gassler
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
| | - Julia A. Vorholt
- Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland
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16
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Barreiro C, Albillos SM, García-Estrada C. Penicillium chrysogenum: Beyond the penicillin. ADVANCES IN APPLIED MICROBIOLOGY 2024; 127:143-221. [PMID: 38763527 DOI: 10.1016/bs.aambs.2024.02.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2024]
Abstract
Almost one century after the Sir Alexander Fleming's fortuitous discovery of penicillin and the identification of the fungal producer as Penicillium notatum, later Penicillium chrysogenum (currently reidentified as Penicillium rubens), the molecular mechanisms behind the massive production of penicillin titers by industrial strains could be considered almost fully characterized. However, this filamentous fungus is not only circumscribed to penicillin, and instead, it seems to be full of surprises, thereby producing important metabolites and providing expanded biotechnological applications. This review, in addition to summarizing the classical role of P. chrysogenum as penicillin producer, highlights its ability to generate an array of additional bioactive secondary metabolites and enzymes, together with the use of this microorganism in relevant biotechnological processes, such as bioremediation, biocontrol, production of bioactive nanoparticles and compounds with pharmaceutical interest, revalorization of agricultural and food-derived wastes or the enhancement of food industrial processes and the agricultural production.
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Affiliation(s)
- Carlos Barreiro
- Área de Bioquímica y Biología Molecular, Departamento de Biología Molecular, Facultad de Veterinaria, Universidad de León, León, Spain; Instituto de Biología Molecular, Genómica y Proteómica (INBIOMIC), Universidad de León, León, Spain.
| | - Silvia M Albillos
- Área de Bioquímica y Biología Molecular, Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Burgos, Spain
| | - Carlos García-Estrada
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, León, Spain; Instituto de Biomedicina (IBIOMED), Universidad de León, León, Spain
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17
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Yi D, Mertz JE. Paul Berg and the origins of recombinant DNA. Cell 2024; 187:1019-1023. [PMID: 38428385 DOI: 10.1016/j.cell.2024.01.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Revised: 12/29/2023] [Accepted: 01/03/2024] [Indexed: 03/03/2024]
Abstract
In fall 1972, Paul Berg's laboratory published articles in PNAS describing two methods for constructing recombinant DNAs in vitro. He received half of the 1980 Nobel Prize in Chemistry for this landmark accomplishment. Here, we describe how this discovery came about, revolutionizing both biological research and the pharmaceutical industry.
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Affiliation(s)
- Doogab Yi
- Seoul National University, Seoul, South Korea
| | - Janet E Mertz
- University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA.
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18
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Hanai R, Hosono K. Screening for termination sequences of a rolling-circle plasmid: a novel scheme using genomic DNA. J GEN APPL MICROBIOL 2024; 69:196-205. [PMID: 37081609 DOI: 10.2323/jgam.2023.04.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/22/2023]
Abstract
The Escherichia coli genome was searched for potential terminators of the rolling-circle replication of staphylococcal plasmid pC194. The replication origin of pC194 was randomly inserted into the E. coli chromosome and rolling-circle replication was initiated by producing pC194's replication protein from a plasmid. Circular DNA resulting from termination in the chromosome was recovered from 42 of the 100 insertion clones screened. The nucleotide sequences at the ends of the chromosomal segment in the recovered DNA were determined and used to identify the locus of integration and the point of termination. The sequence beyond the termination point was retrieved from the database. This information would have been unrecoverable if synthetic random sequences had been used for screening. The consensus sequence based on the discovered potential terminators was consistent with the results of previous and new experiments. The recovered circular DNAs contain a hybrid origin consisting of a 5' part derived from the chromosomal DNA and a 3' part of the integrated origin. Two such hybrid origins were examined for initiation function and shown to be as effective as the authentic pC194 origin. These results suggest a possible evolutionary mechanism in which a rolling-circle plasmid may acquire genes from the host organism.
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Affiliation(s)
- Ryo Hanai
- Department of Life Science and Research Center for Life Science, College of Science, Rikkyo University
| | - Kazuya Hosono
- Department of Life Science and Research Center for Life Science, College of Science, Rikkyo University
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19
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NAGATA S. Cloning of human Type I interferon cDNAs. PROCEEDINGS OF THE JAPAN ACADEMY. SERIES B, PHYSICAL AND BIOLOGICAL SCIENCES 2024; 100:1-14. [PMID: 37648466 PMCID: PMC10864172 DOI: 10.2183/pjab.100.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Accepted: 05/29/2023] [Indexed: 09/01/2023]
Abstract
In the late 1970s, crude interferon samples were found to exhibit anti-tumour activity. This discovery led to the interferon as a "magic drug" for cancer patients. Many groups, including those in Tokyo, Zürich, and San Francisco, attempted to identify human interferon cDNAs. Tadatsugu Taniguchi was the first to announce the cloning of human interferon-β cDNA in the December 1979 issue of Proc. Jpn. Acad. Ser. B. This was followed by the cloning of human interferon-α by a Zürich group and interferon-γ by a group in Genentech in San Francisco. Recombinant interferon proteins were produced on a large scale, and interferon-α was widely used to treat C-type hepatitis patients. The biological functions of interferons were quickly elucidated with the purified recombinant interferons. The molecular mechanisms underlying virus-induced interferon gene expression were also examined using cloned chromosomal genes. The background that led to interferon gene cloning and its impact on cytokine gene hunting is described herein.
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Affiliation(s)
- Shigekazu NAGATA
- Biochemistry & Immunology, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan
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20
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Wu J, Zhang M, Huang J, Guan J, Hu C, Shi M, Hu S, Wang S, Ma H. Enhanced absorbance detection system for online bacterial monitoring in digital microfluidics. Analyst 2023; 148:4659-4667. [PMID: 37615041 DOI: 10.1039/d3an01049j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/25/2023]
Abstract
We report a fully integrated digital microfluidic absorbance detection system with an enhanced sensitivity for online bacterial monitoring. Through a 100 μm gap in the chip, our optical detection system has a detection sensitivity for a BCA protein concentration of 0.1 mg mL-1. The absorbance detection limit of our system is 1.4 × 10-3 OD units, which is one order of magnitude better than that of the existing studies. The system's linear region is 0.1-7 mg mL-1, and the dynamic range is 0-25 mg mL-1. We measured the growth curves of wild-type and E. coli transformed with resistance plasmids and mixed at different ratios on chip. We sorted out the bacterial species including highly viable single cells based on the difference in absorbance data of growth curves. We explored the changes in the growth curves of E. coli under different concentrations of resistant media. In addition, we successfully screened for the optimal growth environment of the bacteria, in which the growth rate of PET30a-DH5α (in a medium with 33 μg mL-1 kanamycin resistance) was significantly higher than that of a 1 mg mL-1 resistance medium. In conclusion, the enhanced digital microfluidic absorbance detection system exhibits exceptional sensitivity, enabling precise bacterial monitoring and growth curve analysis, while also laying the foundation for DMF-based automated bioresearch platforms, thus advancing research in the life sciences.
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Affiliation(s)
- Jingya Wu
- Guangdong-Hong Kong-Macao Joint Laboratory for Intelligent Micro-Nano Optoelectronic Technology, School of Physics and Optoelectronic Engineering, Foshan University, Foshan 528225, P. R. China.
| | - Maolin Zhang
- School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, Anhui, P. R. China
- CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, P. R. China.
| | - Jianle Huang
- Guangdong ACXEL Micro & Nano Tech Co., Ltd, Guangdong Province, 528000, P. R. China
| | - Jingxin Guan
- Guangdong ACXEL Micro & Nano Tech Co., Ltd, Guangdong Province, 528000, P. R. China
| | - Chenxuan Hu
- School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, Anhui, P. R. China
- CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, P. R. China.
| | - Mude Shi
- Guangdong ACXEL Micro & Nano Tech Co., Ltd, Guangdong Province, 528000, P. R. China
| | - Siyi Hu
- CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, P. R. China.
| | - Shurong Wang
- Guangdong-Hong Kong-Macao Joint Laboratory for Intelligent Micro-Nano Optoelectronic Technology, School of Physics and Optoelectronic Engineering, Foshan University, Foshan 528225, P. R. China.
| | - Hanbin Ma
- CAS Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, P. R. China.
- Guangdong ACXEL Micro & Nano Tech Co., Ltd, Guangdong Province, 528000, P. R. China
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21
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Fisher DJ, Beare PA. Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria. Front Cell Infect Microbiol 2023; 13:1202245. [PMID: 37404720 PMCID: PMC10315504 DOI: 10.3389/fcimb.2023.1202245] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Accepted: 05/22/2023] [Indexed: 07/06/2023] Open
Abstract
The ability to genetically manipulate a pathogen is fundamental to discovering factors governing host-pathogen interactions at the molecular level and is critical for devising treatment and prevention strategies. While the genetic "toolbox" for many important bacterial pathogens is extensive, approaches for modifying obligate intracellular bacterial pathogens were classically limited due in part to the uniqueness of their obligatory lifestyles. Many researchers have confronted these challenges over the past two and a half decades leading to the development of multiple approaches to construct plasmid-bearing recombinant strains and chromosomal gene inactivation and deletion mutants, along with gene-silencing methods enabling the study of essential genes. This review will highlight seminal genetic achievements and recent developments (past 5 years) for Anaplasma spp., Rickettsia spp., Chlamydia spp., and Coxiella burnetii including progress being made for the still intractable Orientia tsutsugamushi. Alongside commentary of the strengths and weaknesses of the various approaches, future research directions will be discussed to include methods for C. burnetii that should have utility in the other obligate intracellular bacteria. Collectively, the future appears bright for unraveling the molecular pathogenic mechanisms of these significant pathogens.
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Affiliation(s)
- Derek J. Fisher
- School of Biological Sciences, Southern Illinois University, Carbondale, IL, United States
| | - Paul A. Beare
- Rocky Mountain Laboratory, National Institute of Health, Hamilton, MT, United States
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22
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Xiang W, Hong S, Xue Y, Ma Y. Functional Analysis of Novel alkB Genes Encoding Long-Chain n-Alkane Hydroxylases in Rhodococcus sp. Strain CH91. Microorganisms 2023; 11:1537. [PMID: 37375039 DOI: 10.3390/microorganisms11061537] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 06/05/2023] [Accepted: 06/07/2023] [Indexed: 06/29/2023] Open
Abstract
Rhodococcus sp. strain CH91 is capable of utilizing long-chain n-alkanes as the sole carbon source. Two new genes (alkB1 and alkB2) encoding AlkB-type alkane hydroxylase were predicted by its whole-genome sequence analysis. The purpose of this study was to elucidate the functional role of alkB1 and alkB2 genes in the n-alkane degradation of strain CH91. RT-qPCR analyses revealed that the two genes were induced by n-alkanes ranging from C16 to C36 and the expression of the alkB2 gene was up-regulated much higher than that of alkB1. The knockout of the alkB1 or alkB2 gene in strain CH91 resulted in the obvious reduction of growth and degradation rates on C16-C36 n-alkanes and the alkB2 knockout mutant exhibited lower growth and degradation rate than the alkB1 knockout mutant. When gene alkB1 or alkB2 was heterologously expressed in Pseudomonas fluorescens KOB2Δ1, the two genes could restore its alkane degradation activity. These results demonstrated that both alkB1 and alkB2 genes were responsible for C16-C36 n-alkanes' degradation of strain CH91, and alkB2 plays a more important role than alkB1. The functional characteristics of the two alkB genes in the degradation of a broad range of n-alkanes make them potential gene candidates for engineering the bacteria used for bioremediation of petroleum hydrocarbon contaminations.
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Affiliation(s)
- Wei Xiang
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Shan Hong
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yanfen Xue
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yanhe Ma
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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23
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Farooqui AK, Ahmad H, Rehmani MU, Husain A. Quick and easy method for extraction and purification of Pfu-Sso7d, a high processivity DNA polymerase. Protein Expr Purif 2023; 208-209:106276. [PMID: 37156451 DOI: 10.1016/j.pep.2023.106276] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Revised: 04/19/2023] [Accepted: 04/20/2023] [Indexed: 05/10/2023]
Abstract
The polymerase chain reaction is an extensively used technique with numerous applications in the field of biological sciences. In addition to naturally occurring DNA polymerases with varying processivity and fidelity, genetically engineered recombinant DNA polymerases are also used in PCR. The Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA binding protein, to the polymerase domain of the Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity. Expensive commercial variants of Pfu-Sso7d are sold under various trade names. Here, we report a quick, cost and time-efficient purification protocol and an optimized buffer system for Pfu-Sso7d. We evaluated precipitation efficiencies of varying concentrations of ethanol and acetone and compared the activities of the precipitated enzyme. Although both the solvents efficiently precipitated Pfu-Sso7d, acetone showed better precipitation efficiency. Purified Pfu-Sso7d showed excellent activities in the PCR of templates with varying lengths and GC contents. We also report a buffer system that works with Pfu-Sso7d as efficiently as commercially available buffers. This quick and efficient purification scheme and buffer system will provide researchers cost-efficient access to fusion polymerases.
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Affiliation(s)
- Afreen Kamal Farooqui
- Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India
| | - Haleema Ahmad
- Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India
| | - Mohd Umar Rehmani
- Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India
| | - Afzal Husain
- Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India.
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24
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Campolo N, Mastrogiovanni M, Mariotti M, Issoglio FM, Estrin D, Hägglund P, Grune T, Davies MJ, Bartesaghi S, Radi R. Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation. J Biol Chem 2023; 299:102941. [PMID: 36702251 PMCID: PMC10011836 DOI: 10.1016/j.jbc.2023.102941] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 01/13/2023] [Accepted: 01/16/2023] [Indexed: 01/25/2023] Open
Abstract
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
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Affiliation(s)
- Nicolás Campolo
- Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Mauricio Mastrogiovanni
- Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Michele Mariotti
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Federico M Issoglio
- CONICET-Universidad de Buenos Aires, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Buenos Aires, Argentina; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Oeiras, Portugal
| | - Darío Estrin
- CONICET-Universidad de Buenos Aires, Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE), Buenos Aires, Argentina; Departamento de Química Inorgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Analítica y Química Física, Buenos Aires, Argentina
| | - Per Hägglund
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Tilman Grune
- Department of Molecular Toxicology, German Institute of Human Nutrition, Potsdam-Rehbrücke, Nuthetal, Germany; German Center for Cardiovascular Research (DZHK), Berlin, Germany; Department of Physiological Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria
| | - Michael J Davies
- Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark
| | - Silvina Bartesaghi
- Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay
| | - Rafael Radi
- Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
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25
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Cloning and expression of staphylokinase-streptokinase recombinant protein in E. coli BL21(DE3). Biologia (Bratisl) 2023. [DOI: 10.1007/s11756-023-01311-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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26
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Sheridan PO, Odat MA, Scott KP. Establishing genetic manipulation for novel strains of human gut bacteria. MICROBIOME RESEARCH REPORTS 2023; 2:1. [PMID: 38059211 PMCID: PMC10696588 DOI: 10.20517/mrr.2022.13] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 10/29/2022] [Accepted: 12/12/2022] [Indexed: 12/08/2023]
Abstract
Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as Bifidobacterium, Bacteroides and Roseburia. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota.
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Affiliation(s)
- Paul O. Sheridan
- School of Biological and Chemical Sciences, University of Galway, Galway H91 TK33, Ireland
| | - Ma’en Al Odat
- Gut Health Group, Rowett Institute, University of Aberdeen, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK
| | - Karen P. Scott
- Gut Health Group, Rowett Institute, University of Aberdeen, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK
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27
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Liu D, Siguenza NE, Zarrinpar A, Ding Y. Methods of DNA introduction for the engineering of commensal microbes. ENGINEERING MICROBIOLOGY 2022; 2:100048. [PMID: 39628703 PMCID: PMC11610962 DOI: 10.1016/j.engmic.2022.100048] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 08/19/2022] [Accepted: 08/29/2022] [Indexed: 12/06/2024]
Abstract
The microbiome is an essential component of ecological systems and is comprised of a diverse array of microbes. Over the past decades, the accumulated observational evidence reveals a close correlation between the microbiome and human health and disease. Many groups are now manipulating individual microbial strains, species and the community as a whole to gain a mechanistic understanding of the functions of the microbiome. Here, we discuss three major approaches for introducing DNA to engineer model bacteria and isolated undomesticated bacteria, including transformation, transduction, and conjugation. We provide an overview of these approaches and describe the advantages and limitations of each method. In addition, we highlight examples of human microbiome engineering using these approaches. Finally, we provide perspectives for the future of microbiome engineering.
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Affiliation(s)
- Dake Liu
- Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, University of Florida, Gainesville 32610, Florida, United States
| | - Nicole E. Siguenza
- Division of Gastroenterology, Center for Microbiome Innovation, University of California, La Jolla, San Diego 92093, California , United States
| | - Amir Zarrinpar
- Division of Gastroenterology, Center for Microbiome Innovation, University of California, La Jolla, San Diego 92093, California , United States
- VA San Diego Health System, La Jolla 92161, California, United States
| | - Yousong Ding
- Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, University of Florida, Gainesville 32610, Florida, United States
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28
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Past, Present, and Future of Genome Modification in Escherichia coli. Microorganisms 2022; 10:microorganisms10091835. [PMID: 36144436 PMCID: PMC9504249 DOI: 10.3390/microorganisms10091835] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2022] [Revised: 09/05/2022] [Accepted: 09/05/2022] [Indexed: 12/04/2022] Open
Abstract
Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.
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29
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A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide. Sci Rep 2022; 12:11629. [PMID: 35804085 PMCID: PMC9270391 DOI: 10.1038/s41598-022-15997-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Accepted: 07/04/2022] [Indexed: 11/09/2022] Open
Abstract
The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA-protein complexes into clinically important bacteria.
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30
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Barela Hudgell MA, Smith LC. Lipofection mediated transfection fails for sea urchin coelomocytes. PLoS One 2022; 17:e0267911. [PMID: 35522665 PMCID: PMC9075664 DOI: 10.1371/journal.pone.0267911] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Accepted: 04/19/2022] [Indexed: 11/19/2022] Open
Abstract
Molecular cloning, gene manipulation, gene expression, protein function, and gene regulation all depend on the introduction of nucleic acids into target cells. Multiple methods have been developed to facilitate such delivery including instrument based microinjection and electroporation, biological methods such as transduction, and chemical methods such as calcium phosphate precipitation, cationic polymers, and lipid based transfection, also known as lipofection. Here we report attempts to lipofect sea urchin coelomocytes using DOTAP lipofection reagent packaged with a range of molecules including fluorochromes, in addition to expression constructs, amplicons, and RNA encoding GFP. DOTAP has low cytotoxicity for coelomocytes, however, lipofection of a variety of molecules fails to produce any signature of success based on results from fluorescence microscopy and flow cytometry. While these results are negative, it is important to report failed attempts so that others conducting similar research do not repeat these approaches. Failure may be the outcome of elevated ionic strength of the coelomocyte culture medium, uptake and degradation of lipoplexes in the endosomal-lysosomal system, failure of the nucleic acids to escape the endosomal vesicles and enter the cytoplasm, and difficulties in lipofecting primary cultures of phagocytic cells. We encourage others to build on this report by using our information to optimize lipofection with a range of other approaches to work towards establishing a successful method of transfecting adult cells from marine invertebrates.
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Affiliation(s)
- Megan A. Barela Hudgell
- Department of Biological Sciences, George Washington University, Washington, DC, United States of America
| | - L. Courtney Smith
- Department of Biological Sciences, George Washington University, Washington, DC, United States of America
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31
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Epova EY, Shevelev AB, Shurubor YI, Cooper AJL, Biryukova YK, Bogdanova ES, Tyno YY, Lebedeva AA, Krasnikov BF. A novel efficient producer of human ω-amidase (Nit2) in Escherichia coli. Anal Biochem 2021; 632:114332. [PMID: 34391728 DOI: 10.1016/j.ab.2021.114332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Revised: 07/09/2021] [Accepted: 08/06/2021] [Indexed: 11/25/2022]
Abstract
Nit2/ω-amidase catalyzes the hydrolysis of α-ketoglutaramate (KGM, the α-keto acid analogue of glutamine) to α-ketoglutarate and ammonia. The enzyme also catalyzes the amide hydrolysis of monoamides of 4- and 5-C-dicarboxylates, including α-ketosuccinamate (KSM, the α-keto acid analogue of asparagine) and succinamate (SM). Here we describe an inexpensive procedure for high-yield expression of human Nit2 (hNit2) in Escherichia coli and purification of the expressed protein. This work includes: 1) the design of a genetic construct (pQE-Nit22) obtained from the previously described construct (pQE-Nit2) by replacing rare codons within an 81 bp-long DNA fragment "preferred" by E. coli near the translation initiation site; 2) methods for producing and maintaining the pQE-Nit22 construct; 3) purification of recombinant hNit2; and 4) activity measurements of the purified enzyme with KGM and SM. Important features of the hNit2 gene within the pQE-Nit22 construct are: 1) optimized codon composition, 2) the presence of an N-terminus His6 tag immediately after the initiating codon ATG (Met) that permits efficient purification of the end-product on a Ni-NTA-agarose column. We anticipate that the availability of high yield hNit2/ω-amidase will be helpful in elucidating the normal and pathological roles of this enzyme and in the design of specific inhibitors.
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Affiliation(s)
- Ekaterina Yu Epova
- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia
| | - Alexei B Shevelev
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | | | - Arthur J L Cooper
- Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595, USA
| | - Yulia K Biryukova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | - Elena S Bogdanova
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia; Plekhanov Russian University of Economics, Moscow, Russia
| | - Yaroslav Ya Tyno
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | - Anna A Lebedeva
- Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
| | - Boris F Krasnikov
- Centre for Strategic Planning of FMBA of the Russian Federation, Moscow, Russia; Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595, USA.
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32
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Green MR, Sambrook J. Cloning and Transformation with Plasmid Vectors. Cold Spring Harb Protoc 2021; 2021:2021/11/pdb.top101170. [PMID: 34725175 DOI: 10.1101/pdb.top101170] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Plasmids occupy a place of honor in molecular cloning: They were used in the first recombinant DNA experiments and, 40 or more years later, they remain as the carriage horses of molecular cloning. After almost half a century of sequential improvement in design, today's plasmid vectors are available in huge variety, are often optimized for specific purposes, and bear only passing resemblance to their forebears. Here, various features of plasmid vectors and methods for transforming E. coli cells are introduced.
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33
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Dokka N, Mahajan MM, Sahu B, Marathe A, Singh HK, Sivalingam PN. Molecular analysis, infectivity and host range of Tomato leaf curl Karnataka virus associated with Corchorus yellow vein mosaic betasatellite. Virus Res 2021; 303:198521. [PMID: 34314770 DOI: 10.1016/j.virusres.2021.198521] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 07/16/2021] [Accepted: 07/21/2021] [Indexed: 11/21/2022]
Abstract
Severe leaf curl disease of tomato (ToLCD) was noticed recently in the central parts of India and is an emerging threat to the cultivation of tomato. The genomic components of the begomovirus isolate, DNA A and betasatellite associated with ToLCD were cloned by rolling circle amplification method and sequenced. The sequence analysis revealed that the DNA A (2766 nt) of this isolate had the nucleotide identity of >91% with other strains of Tomato leaf curl Karnataka virus (ToLCKV), hence this isolate is proposed as a strain of ToLCKV, named as ToLCKV-Raipur. Similarly, the betasatellite molecule (1355 nt) had the highest identity of 91.1% with Corchorus yellow vein mosaic betasatellite (CoYVMB) and named as CoYVMB-Raipur. The full-length dimerized clones of these two genomic components were agroinoculated on natural (tomato), experimental (Nicotiana benthamiana) hosts and other 20 plant species belong to six different families. The severe leaf curl symptoms appeared only in the hosts, N. benthamiana, and in tomato inoculated with ToLCKV-Raipur alone and ToLCKV-Raipur with CoYVMB-Raipur after 8 and 16-18 days inoculation, respectively. This isolate was also transmissible to healthy tomato plants by whitefly from the tomato plant agroinoculated with ToLCKV-Raipur alone and with CoYVMB-Raipur and produced symptoms within 14-16 days after inoculation. Interestingly, this isolate infects horse gram and chilli by whitefly transmission and both the hosts showed positive for DNA A alone but not for betasatellite. Quantification of the genomic components of this isolate with the agroinoculated N. benthamiana samples by qRT-PCR results showed that the quantity of ToLCKV-Raipur was enhanced by three-fold while inoculated with CoYVMB-Raipur compared to ToLCKV-Raipur alone inoculated plants. However, CoYVMB-Raipur did not enhance the levels of ToLCKV-Raipur in the agroinoculated tomato plants. This is the first evidence of the natural co-occurrence of ToLCKV with betasatellite, CoYVMB causing ToLCD.
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Affiliation(s)
- Narasimham Dokka
- ICAR-National Institute of Biotic Stress Management, Baronda, Raipur, Chhattisgarh 493225, India
| | - Mahesh Mohanrao Mahajan
- ICAR-National Institute of Biotic Stress Management, Baronda, Raipur, Chhattisgarh 493225, India
| | - Bhimeshwari Sahu
- ICAR-National Institute of Biotic Stress Management, Baronda, Raipur, Chhattisgarh 493225, India
| | - Ashish Marathe
- ICAR-National Institute of Biotic Stress Management, Baronda, Raipur, Chhattisgarh 493225, India
| | - Harvinder Kumar Singh
- Department of Plant Pathology, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh 492012, India
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Bacillus pumilus 15.1, a Strain Active against Ceratitis capitata, Contains a Novel Phage and a Phage-Related Particle with Bacteriocin Activity. Int J Mol Sci 2021; 22:ijms22158164. [PMID: 34360927 PMCID: PMC8347963 DOI: 10.3390/ijms22158164] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 07/19/2021] [Accepted: 07/26/2021] [Indexed: 11/16/2022] Open
Abstract
A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata, the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus, respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.
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35
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Hartz P, Strohmaier SJ, El-Gayar BM, Abdulmughni A, Hutter MC, Hannemann F, Gillam EMJ, Bernhardt R. Resurrection and characterization of ancestral CYP11A1 enzymes. FEBS J 2021; 288:6510-6527. [PMID: 34092040 DOI: 10.1111/febs.16054] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 05/22/2021] [Accepted: 06/04/2021] [Indexed: 01/16/2023]
Abstract
Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A_Mammal_N101 and CYP11A_N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A_N1 showed increased thermostability (~ 25 °C increase in T50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A_N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals.
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Affiliation(s)
- Philip Hartz
- Department of Biochemistry, Saarland University, Saarbrücken, Germany
| | - Silja J Strohmaier
- School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Australia
| | - Basma M El-Gayar
- Department of Biochemistry, Saarland University, Saarbrücken, Germany
| | - Ammar Abdulmughni
- Department of Biochemistry, Saarland University, Saarbrücken, Germany
| | - Michael C Hutter
- Center for Bioinformatics, Saarland University, Saarbrücken, Germany
| | - Frank Hannemann
- Department of Biochemistry, Saarland University, Saarbrücken, Germany
| | - Elizabeth M J Gillam
- School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Australia
| | - Rita Bernhardt
- Department of Biochemistry, Saarland University, Saarbrücken, Germany
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36
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Mahmud Z, Shabnam SA, Mishu ID, Johura FT, Mannan SB, Sadique A, Islam LN, Alam M. Virotyping, genotyping, and molecular characterization of multidrug resistant Escherichia coli isolated from diarrheal patients of Bangladesh. GENE REPORTS 2021. [DOI: 10.1016/j.genrep.2021.101182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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37
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Wang Y, Sun S, Yu L, Hu S, Fan W, Leng F, Ma J. Optimization and mechanism exploration for Escherichia coli transformed with plasmid pUC19 by the combination with ultrasound treatment and chemical method. ULTRASONICS SONOCHEMISTRY 2021; 74:105552. [PMID: 33887660 PMCID: PMC8091046 DOI: 10.1016/j.ultsonch.2021.105552] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/06/2020] [Revised: 03/28/2021] [Accepted: 04/06/2021] [Indexed: 06/12/2023]
Abstract
As a basic technique of molecular cloning, bio-transformation has been successfully used in the fields of biomedicine and food processing. In this study, we established a transformation system of exogenous DNA into E. coli cells mediated by ultrasound. Under the optimal conditions (i.e. 35 °C, 40 W, 25 s, OD600 = 0.4-0.6) optimized by RSM, the transformation efficiency reached at 1.006 × 107 CFU/μg DNA. The results of membrane permeability, macromolecular substance and cell structure analysis before and after ultrasound treatment showed that the damage of host cells induced by lower (40 W) ultrasound and shorter ultrasound time (25 s) was reversible, and the transformation efficiency and cell survival rate were not significantly affected under this condition. In brief, proper changes in cell membrane and cell wall were the basic conditions for host cells to uptake exogenous DNA, while, whether exogenous DNA could be replicated and expressed in cells depends on the viability of host cells.
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Affiliation(s)
- Yonggang Wang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
| | - Shangchen Sun
- School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050, China
| | - Linmiao Yu
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
| | - Shu Hu
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
| | - Wenguang Fan
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China
| | - Feifan Leng
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
| | - Jianzhong Ma
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China.
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Schultenkämper K, Gütle DD, López MG, Keller LB, Zhang L, Einsle O, Jacquot JP, Wendisch VF. Interrogating the Role of the Two Distinct Fructose-Bisphosphate Aldolases of Bacillus methanolicus by Site-Directed Mutagenesis of Key Amino Acids and Gene Repression by CRISPR Interference. Front Microbiol 2021; 12:669220. [PMID: 33995334 PMCID: PMC8119897 DOI: 10.3389/fmicb.2021.669220] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Accepted: 03/30/2021] [Indexed: 12/13/2022] Open
Abstract
The Gram-positive Bacillus methanolicus shows plasmid-dependent methylotrophy. This facultative ribulose monophosphate (RuMP) cycle methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBAC is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBAP is found on the natural plasmid pBM19 and is induced during methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBAC and FBAP differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBAP. However, both FBAC and FBAP maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBAC in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBAC and FBAP concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fba C RNA levels were reduced by CRISPRi, but concomitantly the fba P RNA levels were increased. Vice versa, a similar compensatory increase of the fba C RNA levels was observed when fba P was repressed by CRISPRi. In addition, targeting fba P decreased tkt P RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tkt P RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tkt C.
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Affiliation(s)
- Kerstin Schultenkämper
- Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany
| | | | - Marina Gil López
- Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany
| | - Laura B Keller
- Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany
| | - Lin Zhang
- Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Freiburg, Germany
| | - Oliver Einsle
- Institute for Biochemistry, Albert-Ludwigs-University Freiburg, Freiburg, Germany
| | | | - Volker F Wendisch
- Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Bielefeld, Germany
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McGinn J, Lamason RL. The enigmatic biology of rickettsiae: recent advances, open questions and outlook. Pathog Dis 2021; 79:ftab019. [PMID: 33784388 PMCID: PMC8035066 DOI: 10.1093/femspd/ftab019] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 03/26/2021] [Indexed: 02/05/2023] Open
Abstract
Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses and are among the oldest known vector-borne pathogens. Members of this genus are extraordinarily diverse and exhibit a broad host range. To establish intracellular infection, Rickettsia species undergo complex, multistep life cycles that are encoded by heavily streamlined genomes. As a result of reductive genome evolution, rickettsiae are exquisitely tailored to their host cell environment but cannot survive extracellularly. This host-cell dependence makes for a compelling system to uncover novel host-pathogen biology, but it has also hindered experimental progress. Consequently, the molecular details of rickettsial biology and pathogenesis remain poorly understood. With recent advances in molecular biology and genetics, the field is poised to start unraveling the molecular mechanisms of these host-pathogen interactions. Here, we review recent discoveries that have shed light on key aspects of rickettsial biology. These studies have revealed that rickettsiae subvert host cells using mechanisms that are distinct from other better-studied pathogens, underscoring the great potential of the Rickettsia genus for revealing novel biology. We also highlight several open questions as promising areas for future study and discuss the path toward solving the fundamental mysteries of this neglected and emerging human pathogen.
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Affiliation(s)
- Jon McGinn
- Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
| | - Rebecca L Lamason
- Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States
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Hartz P, Gehl M, König L, Bernhardt R, Hannemann F. Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium. J Biotechnol 2021; 329:170-179. [PMID: 33600891 DOI: 10.1016/j.jbiotec.2021.02.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 01/11/2021] [Accepted: 02/10/2021] [Indexed: 12/26/2022]
Abstract
Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.
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Affiliation(s)
- Philip Hartz
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Manuel Gehl
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany; Present address: Microbial Protein Structure Group, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany
| | - Lisa König
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Rita Bernhardt
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Frank Hannemann
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany.
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Riley LA, Guss AM. Approaches to genetic tool development for rapid domestication of non-model microorganisms. BIOTECHNOLOGY FOR BIOFUELS 2021; 14:30. [PMID: 33494801 PMCID: PMC7830746 DOI: 10.1186/s13068-020-01872-z] [Citation(s) in RCA: 62] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Accepted: 12/30/2020] [Indexed: 05/04/2023]
Abstract
Non-model microorganisms often possess complex phenotypes that could be important for the future of biofuel and chemical production. They have received significant interest the last several years, but advancement is still slow due to the lack of a robust genetic toolbox in most organisms. Typically, "domestication" of a new non-model microorganism has been done on an ad hoc basis, and historically, it can take years to develop transformation and basic genetic tools. Here, we review the barriers and solutions to rapid development of genetic transformation tools in new hosts, with a major focus on Restriction-Modification systems, which are a well-known and significant barrier to efficient transformation. We further explore the tools and approaches used for efficient gene deletion, DNA insertion, and heterologous gene expression. Finally, more advanced and high-throughput tools are now being developed in diverse non-model microbes, paving the way for rapid and multiplexed genome engineering for biotechnology.
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Affiliation(s)
- Lauren A Riley
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA
- Bredesen Center, University of Tennessee, Knoxville, TN, 37996, USA
| | - Adam M Guss
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.
- Bredesen Center, University of Tennessee, Knoxville, TN, 37996, USA.
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Ji Y, Guan F, Zhou X, Liu X, Wu N, Liu D, Tian J. Construction of a mApple-D6A3-mediated biosensor for detection of heavy metal ions. AMB Express 2020; 10:213. [PMID: 33284386 PMCID: PMC7721944 DOI: 10.1186/s13568-020-01154-9] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2020] [Accepted: 11/27/2020] [Indexed: 11/23/2022] Open
Abstract
Pollution of heavy metals in agricultural environments is a growing problem to the health of the world’s human population. Green, low-cost, and efficient detection methods can help control such pollution. In this study, a protein biosensor, mApple-D6A3, was built from rice-derived Cd2+-binding protein D6A3 fused with the red fluorescent protein mApple at the N-terminus to detect the contents of heavy metals. Fluorescence intensity of mApple fused with D6A3 indicated the biosensor’s sensitivity to metal ions and its intensity was more stable under alkaline conditions. mApple-D6A3 was most sensitive to Cu2+, then Ni2+, then Cd2+. Isothermal titration calorimetry experiments demonstrated that mApple-D6A3 successfully bound to each of these three metal ions, and its ability to bind the ions was, from strongest to weakest, Cu2+ > Cd2+ > Ni2+. There were strong linear relationships between the fluorescence intensity of mApple-D6A3 and concentrations of Cd2+ (0–100 μM), Cu2+ (0–60 μM) and Ni2+ (0–120 μM), and their respective R2 values were 0.994, 0.973 and 0.973. When mApple-D6A3 was applied to detect concentrations of heavy metal ions in water (0–0.1 mM) or culture medium (0–1 mM), its accuracy for detection attained more than 80%. This study demonstrates the potential of this biosensor as a tool for detection of heavy metal ions.
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Ectopic expression of WsMBP1 from Withania somnifera in transgenic tobacco shows insecticidal activity against teak defoliator Hyblaea puera (Lepidoptera: Hyblaeidae). Biologia (Bratisl) 2020. [DOI: 10.2478/s11756-020-00531-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Expression of the rabies virus nucleoprotein and matrix protein in a prokaryotic system at high-levels: An efficacious production method. GENE REPORTS 2020. [DOI: 10.1016/j.genrep.2020.100799] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Higuchi-Takeuchi M, Miyamoto T, Foong CP, Goto M, Morisaki K, Numata K. Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria. Int J Mol Sci 2020; 21:ijms21228625. [PMID: 33207642 PMCID: PMC7697693 DOI: 10.3390/ijms21228625] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Revised: 11/11/2020] [Accepted: 11/14/2020] [Indexed: 11/22/2022] Open
Abstract
Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.
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Affiliation(s)
- Mieko Higuchi-Takeuchi
- Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan; (T.M.); (M.G.); (K.M.)
- Correspondence: (M.H.-T.); (K.N.)
| | - Takaaki Miyamoto
- Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan; (T.M.); (M.G.); (K.M.)
| | - Choon Pin Foong
- Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan;
| | - Mami Goto
- Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan; (T.M.); (M.G.); (K.M.)
| | - Kumiko Morisaki
- Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan; (T.M.); (M.G.); (K.M.)
| | - Keiji Numata
- Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan; (T.M.); (M.G.); (K.M.)
- Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan;
- Correspondence: (M.H.-T.); (K.N.)
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A Reporter System for Fast Quantitative Monitoring of Type 3 Protein Secretion in Enteropathogenic E. coli. Microorganisms 2020; 8:microorganisms8111786. [PMID: 33202599 PMCID: PMC7696366 DOI: 10.3390/microorganisms8111786] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 11/06/2020] [Accepted: 11/09/2020] [Indexed: 12/16/2022] Open
Abstract
The type 3 secretion system is essential for pathogenesis of several human and animal Gram-negative bacterial pathogens. The T3SS comprises a transmembrane injectisome, providing a conduit from the bacterial cytoplasm to the host cell cytoplasm for the direct delivery of effectors (including toxins). Functional studies of T3SS commonly monitor the extracellular secretion of proteins by SDS-PAGE and western blot analysis, which are slow and semi-quantitative in nature. Here, we describe an enzymatic reporter-based quantitative and rapid in vivo assay for T3SS secretion studies in enteropathogenic E. coli (EPEC). The assay monitors the secretion of the fusion protein SctA-PhoA through the injectisome based on a colorimetric assay that quantifies the activity of alkaline phosphatase. We validated the usage of this reporter system by following the secretion in the absence of various injectisome components, including domains of the gatekeeper essential for T3SS function. This platform can now be used for the isolation of mutations, functional analysis and anti-virulence compound screening.
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Shawki A, Ramirez R, Spalinger MR, Ruegger PM, Sayoc-Becerra A, Santos AN, Chatterjee P, Canale V, Mitchell JD, Macbeth JC, Gries CM, Tremblay ML, Hsiao A, Borneman J, McCole DF. The autoimmune susceptibility gene, PTPN2, restricts expansion of a novel mouse adherent-invasive E. coli. Gut Microbes 2020; 11:1547-1566. [PMID: 32586195 PMCID: PMC7524159 DOI: 10.1080/19490976.2020.1775538] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2020] [Revised: 04/30/2020] [Accepted: 05/18/2020] [Indexed: 02/06/2023] Open
Abstract
Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.
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Affiliation(s)
- Ali Shawki
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Rocio Ramirez
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Marianne R. Spalinger
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Paul M. Ruegger
- Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, California, USA
| | - Anica Sayoc-Becerra
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Alina N. Santos
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Pritha Chatterjee
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Vinicius Canale
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | - Jonathan D. Mitchell
- Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, California, USA
| | - John C. Macbeth
- Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, California, USA
| | - Casey M. Gries
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
| | | | - Ansel Hsiao
- Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, California, USA
| | - James Borneman
- Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, California, USA
| | - Declan F. McCole
- Division of Biomedical Sciences, University of California Riverside, Riverside, California, USA
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Abstract
Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry. Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. Intriguingly, the only substrates known to support B. methanolicus growth as sole sources of carbon and energy are methanol, mannitol, and, to a lesser extent, glucose and arabitol. Because fluxomics provides the most direct readout of the cellular phenotype, we hypothesized that comparing methylotrophic and nonmethylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. In this study, we designed and performed a 13C metabolic flux analysis (13C-MFA) of the facultative methylotroph B. methanolicus MGA3 growing on methanol, mannitol, and arabitol to compare the associated metabolic states. On methanol, results showed a greater flux in the ribulose monophosphate (RuMP) pathway than in the tricarboxylic acid (TCA) cycle, thus validating previous findings on the methylotrophy of B. methanolicus. New insights related to the utilization of cyclic RuMP versus linear dissimilation pathways and between the RuMP variants were generated. Importantly, we demonstrated that the linear detoxification pathways and the malic enzyme shared with the pentose phosphate pathway have an important role in cofactor regeneration. Finally, we identified, for the first time, the metabolic pathway used to assimilate arabitol. Overall, those data provide a better understanding of this strain under various environmental conditions. IMPORTANCE Methanol is inexpensive, is easy to transport, and can be produced both from renewable and from fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural methylotrophs such as Bacillus methanolicus is critical to understand the origin of their efficient methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories or to design methylotrophic capability in other strains already used by the industry.
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Kaur A, Muthukumarappa T, Kanta P, Banday AZ, Chidananda MK. Cloning of hok gene into anhydrotetracycline inducible pASK75 vector reveals potent antimicrobial effect of 19 amino acid long N-terminal fragment of hok peptide. Microbiol Immunol 2020; 64:737-746. [PMID: 32930410 DOI: 10.1111/1348-0421.12849] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 09/03/2020] [Accepted: 09/09/2020] [Indexed: 10/23/2022]
Abstract
An important toxin-antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti-microbial property of a 19 amino acid (AA) long N-terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector - pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MId C) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error-prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N-terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MId C of aTc in our experiments was 6-fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N-terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.
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Affiliation(s)
- Anit Kaur
- Department of Biochemistry, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India.,Allergy Immunology Unit, Department of Pediatrics, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
| | - Thungapathra Muthukumarappa
- Department of Biochemistry, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
| | - Poonam Kanta
- Department of Biochemistry, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
| | - Aaqib Zaffar Banday
- Allergy Immunology Unit, Department of Pediatrics, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
| | - Mohana Kumari Chidananda
- Department of Biochemistry, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
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Wang Y, Wang X, Yu L, Tian Y, Li S, Leng F, Ma J, Chen J. Effects of Sr 2 + on the preparation of Escherchia coli DH5α competent cells and plasmid transformation. PeerJ 2020. [DOI: 10.7717/peerj.9480] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Bacterial gene transformation used with Escherichia coli as a desired microorganism is one of the important techniques in genetic engineering. In this study, the preparation of E. coli DH5α competent cells treated with SrCl2 and transformation by heat-shock with pUC19 plasmid was optimized by Response Surface Methodology (RSM). Other five E. coli strains including BL21 (DE3), HB-101, JM109, TOP10 and TG1, three different sizes plasmids (pUC19, pET32a, pPIC9k) were used to verify the protocol, respectively. The transformation mechanism was explored by scanning electron microscope combined with energy dispersive spectrometer (SEM-EDS), atomic absorption spectroscopy (AAS) and Fourier-transform infrared spectroscopy (FT-IR). An equation of regression model was obtained, and the ideal parameters were Sr2 + ions of 90 mM, heat-shock time of 90 s and 9 ng of plasmid. Under this conditions, the transformation efficiency could almost reach to 106 CFU/µg DNA. A small change of the cell surface structure has been observed between E. coli DH5α strain and competent cells by abovementioned spectrum technologies, which implied that a strict regulation mechanism involved in the formation of competent cells and transformation of plasmids. An equation of regression model for the competent cells preparation and plasmid transformation could be applied in gene cloning technology
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Affiliation(s)
- Yonggang Wang
- School of Energy and Power Engineering, Lanzhou University of Technology, Lan Zhou, Gansu, China
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
| | - Xinjian Wang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
| | - Linmiao Yu
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
| | - Yuan Tian
- Lhasa National Ecological Research Station, Key Laboratory of Ecosystem Network Observation and Modelling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing, China
| | - Shaowei Li
- Lhasa National Ecological Research Station, Key Laboratory of Ecosystem Network Observation and Modelling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing, China
| | - Feifan Leng
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
| | - Jianzhong Ma
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
| | - Jixiang Chen
- School of Energy and Power Engineering, Lanzhou University of Technology, Lan Zhou, Gansu, China
- School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, Gansu, China
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