• antimicrobials
• bacteriology

• Escherichia coli/genetics
• Escherichia coli/drug effects
• Plasmids/genetics
• Anti-Bacterial Agents/pharmacology
• Escherichia coli Proteins/genetics
• Drug Resistance, Bacterial/genetics
• Microbial Sensitivity Tests
• Gene Dosage
• Gene Deletion
• DNA Copy Number Variations

• 956154 Horizon 2020
• BG22/00150 Beatriz Galindo program, Ministerio de Universidades, Spain

• Escherichia coli
• Thermus aquaticus
• Autoinduction
• Chemically defined medium
• DNA polymerase

• Bioreactors
• Thermus/enzymology
• Thermus/genetics
• Recombinant Proteins/genetics
• Recombinant Proteins/biosynthesis
• Recombinant Proteins/metabolism
• Fermentation
• Taq Polymerase/genetics
• Taq Polymerase/biosynthesis
• Taq Polymerase/metabolism
• Isopropyl Thiogalactoside/metabolism
• Culture Media
• Escherichia coli/genetics
• Escherichia coli/metabolism
• DNA-Directed DNA Polymerase/genetics

• 1/III.5/ HK/2024 Badan Riset dan Inovasi Nasional
• NKB-507/UN2.RST/HKP.05.00/2024 Universitas Indonesia

• DNA cleavage
• mechanism
• nanoplastics
• size
• surface ligand
• toxicity

• DNA Damage/drug effects
• DNA/chemistry
• Microplastics/chemistry
• Microplastics/toxicity
• DNA Cleavage/drug effects
• Hydrogen-Ion Concentration
• Nanostructures/chemistry
• Nanostructures/toxicity
• Humans

• Chaperonin
• GroEL
• RNA binding protein
• RNA turnover
• Ribonuclease (RNase)

• Chaperonin 60/metabolism
• Chaperonin 60/genetics
• Escherichia coli/genetics
• Escherichia coli/metabolism
• Ribonucleases/metabolism
• Ribonucleases/genetics
• Escherichia coli Proteins/metabolism
• Escherichia coli Proteins/genetics

• Escherichia coli K12/genetics

• JSPS KAKENHI

• DNA, Recombinant
• MicroRNAs/genetics
• RNA, Small Interfering/genetics
• Metabolic Clearance Rate
• Technology
• Recombinant Proteins
• Pharmacokinetics

• P30 CA093373 NCI NIH HHS
• T32 GM144303 NIGMS NIH HHS
• R01 CA225958 NCI NIH HHS
• R35 GM140835 NIGMS NIH HHS
• R01 CA253230 NCI NIH HHS

• protein delivery
• immunization
• cancer therapy

• Bacteria/metabolism
• Cytokines
• Humans
• Immunotherapy/methods
• Interferon-gamma/genetics
• Interferon-gamma/metabolism
• Neoplasms/genetics
• Neoplasms/therapy

• Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)
• National Natural Science Foundation of China (National Science Foundation of China)
• National Key R&D Program of China (2019YFE0110400) Key R&D Program of Hunan Province (2021SK2035)
• National Key R&D Program of China (2020YFA0908800) Guangdong Innovation Platform of Translational Research for Cerebrovascular Diseases, and Shenzhen Science and Technology Innovation Committee [Grant Number. JCYJ20190812171820731 and JCYJ20180507182420114]

• Bacillus subtilis
• cell wall maintenance
• glucolipid
• lipoteichoic acid
• sigma factor

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria constitute important factors in defining interactions with the extracellular milieu. Lysobacter sp. XL1 produces OMVs capable of lysing microbial cells due to the presence in their cargo of bacteriolytic protease L5 (AlpB). Although protein L5 has been functionally and biochemically characterized (including aspects of its packing into OMVs), its role in vesicle biogenesis through genetic deletion of alpB had not been studied previously. Here, we have successfully deleted alpB by allelic replacement and show that the alpB deletion mutant produces a significantly lower amount of OMVs that lack bacteriolytic activity and display altered ultrastructural characteristics in relation to the OMVs produced by the wild-type strain. These results confirm that, as previously proposed, protein L5 participates in OMV production through a mechanism that is not yet fully understood.

• Lysobacter sp. XL1
• antimicrobial potency of vesicles
• bacteriolytic protease L5
• biogenesis of vesicles
• deletion in gene alpB

• BL21(DE3) gp2
• affibody
• amber suppression
• genetic code expansion
• growth-decoupled protein production
• noncanonical amino acid

• CRISPR array
• CRISPR-associated transposase
• chromosomal integration
• insertion sequence
• multicopy

• cellobiose synthesis
• co-expression
• phosphorylases
• ribosome binding sites
• synthetic biology
• whole-cell bioconversion

• Bacteria/genetics
• Cloning, Molecular
• Computational Biology/methods
• Databases, Genetic
• Europe
• Genetic Engineering
• Genetic Vectors
• Software
• Web Browser

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

• Antibody fragments
• Antibody purification
• HIV
• Protein production
• Rhamnose-inducible promoter

• Bacteria/genetics
• Bacteria/metabolism
• Fungi/genetics
• Fungi/metabolism
• Genetic Engineering/history
• Genetic Engineering/methods
• Genetic Engineering/trends
• Genetic Vectors
• History, 20th Century
• History, 21st Century
• Synthetic Biology/methods

To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.

• YFP
• copy number estimation
• endogenous plasmid
• expression vector
• synthetic biology

• CRISPR
• bioengineering
• genetic tools
• genome editing
• genome engineering
• transformation

Temperature REMD reveals how local chemical changes can result in markedly differing conformational landscapes for DNA and RNA hairpin loops.

• Conditional replication
• Plasmid curing
• Tetracycline-dependent
• pBR322

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc.

Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α–amylase.

The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

• Tn5
• hrpZ
• mutant non-pathogenic
• phylogenetic
• transmid

• bacterial cell division cycle
• nucleoid complexity and segregation
• peptidoglycan biosynthesis
• size and shape determination
• transertion

• Chronic diseases
• Gastrointestinal diseases
• Liver disease
• Nutritional genomics
• Obesity

• Digestive System Diseases/diagnosis
• Digestive System Diseases/genetics
• Digestive System Diseases/therapy
• Gastroenterology/methods
• Gene-Environment Interaction
• Genetic Markers
• Genetic Predisposition to Disease
• Genetic Testing
• Genomics
• Humans
• Phenotype
• Precision Medicine
• Predictive Value of Tests
• Prognosis
• Risk Factors
• Specialization

Formation of patterns is a common feature in the development of multicellular organism as well as of microbial communities. To investigate the formation of gene expression patterns in colonies, we build a mathematical model of two-dimensional colony growth, where cells carry a coupled positive-and-negative-feedback circuit. We demonstrate that the model can produce sectored, target (concentric), uniform, and scattered expression patterns of regulators, depending on gene expression dynamics and nutrient diffusion. We reconstructed the same regulatory structure in Escherichia coli cells and found gene expression patterns on the surface of colonies similar to the ones produced by the computer simulations. By comparing computer simulations and experimental results, we observed that very simple rules of gene expression can yield a spectrum of well-defined patterns in a growing colony. Our results suggest that variations of the protein content among cells lead to a high level of heterogeneity in colonies.

Importance Formation of patterns is a common feature in the development of microbial communities. In this work, we show that a simple genetic circuit composed of a positive-feedback loop and a negative-feedback loop can produce diverse expression patterns in colonies. We obtained similar sets of gene expression patterns in the simulations and in the experiments. Because the combination of positive feedback and negative feedback is common in intracellular molecular networks, our results suggest that the protein content of cells is highly diversified in colonies.

Formation of patterns is a common feature in the development of microbial communities. In this work, we show that a simple genetic circuit composed of a positive-feedback loop and a negative-feedback loop can produce diverse expression patterns in colonies. We obtained similar sets of gene expression patterns in the simulations and in the experiments. Because the combination of positive feedback and negative feedback is common in intracellular molecular networks, our results suggest that the protein content of cells is highly diversified in colonies.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.

Production of recombinant DNA in bacterial cells is an essential technique in molecular biology. Plasmids are usually maintained in an E. coli host by antibiotic selection. However, there are only a few antibiotic-resistance markers available in common use. Here we report the adoption of a novel selection marker, mfabI (mutant fabI) for plasmid propagation in E. coli. mfabI expands the limited repertoire of selection markers and allows for more efficient molecular manipulation and plasmid propagation in E. coli. We show that mfabI is not only an efficient plasmid selection marker, but it also possesses unique activity that may facilitate molecular manipulation of unstable sequences. Furthermore, we have incorporated mfabI in the recombineering tool kit for generating mouse gene targeting vectors and demonstrate the advantage of using mfabI-containing recombineering vectors.

• Anti-Bacterial Agents/pharmacology
• Cloning, Molecular
• DNA, Recombinant
• Drug Resistance, Microbial/genetics
• Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/genetics
• Escherichia coli/drug effects
• Escherichia coli/genetics
• Escherichia coli/growth & development
• Escherichia coli Proteins/genetics
• Fatty Acid Synthase, Type II/genetics
• Gene Order
• Genetic Markers
• Plasmids/genetics
• Recombination, Genetic
• Transformation, Bacterial
• Triclosan/pharmacology

• R01 GM101974 NIGMS NIH HHS
• R01GM101974 NIGMS NIH HHS

Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2–4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood.

The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2–4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promoter analysis indicated the above regulatory circuits were shared by several other closely related Vibrios but with slight exceptions.

This study gave a comprehensive computational and characterization of the direct transcriptional regulation of five target genes, opaR, qrr2–4 and ahpA, by OpaR in V. parahaemolyticus. These characterized regulatory circuits were conserved in V. harveyi and V. parahaemolyticus.

• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• Binding Sites
• DNA, Bacterial/genetics
• DNA-Binding Proteins/genetics
• DNA-Binding Proteins/metabolism
• Gene Expression Regulation, Bacterial
• Mutation
• Phylogeny
• Promoter Regions, Genetic
• Quorum Sensing
• Regulatory Sequences, Nucleic Acid/genetics
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Vibrio parahaemolyticus/genetics
• Vibrio parahaemolyticus/metabolism

Whole-genome sequencing of bacteria has proceeded at an exponential pace but annotation validation has lagged behind. For instance, the MetJ regulon, which controls methionine biosynthesis and transport, has been studied almost exclusively in E. coli and Salmonella, but homologs of MetJ exist in a variety of other species. These include some that are pathogenic (e.g. Yersinia) and some that are important for environmental remediation (e.g. Shewanella) but many of which have not been extensively characterized in the literature.

We have determined the likely composition of the MetJ regulon in all species which have MetJ homologs using bioinformatics techniques. We show that the core genes known from E. coli are consistently regulated in other species, and we identify previously unknown members of the regulon. These include the cobalamin transporter, btuB; all the genes involved in the methionine salvage pathway; as well as several enzymes and transporters of unknown specificity.

The MetJ regulon is present and functional in five orders of gammaproteobacteria: Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales. New regulatory activity for MetJ was identified in the genomic data and verified experimentally. This strategy should be applicable for the elucidation of regulatory pathways in other systems by using the extensive sequencing data currently being generated.

• Biotechnology
• Escherichia coli/enzymology
• Escherichia coli/genetics
• Escherichia coli/metabolism
• Escherichia coli Proteins/genetics
• Escherichia coli Proteins/metabolism
• Formate Dehydrogenases/genetics
• Formate Dehydrogenases/metabolism
• Hydrogenase/genetics
• Hydrogenase/metabolism
• Mutation
• Nitrate Reductase/genetics
• Nitrate Reductase/metabolism

While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.

Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such "benign" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored.

We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size – all antibiotic-resistant non-producers – was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented.

The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

• Computer Simulation
• Escherichia coli/physiology
• Escherichia coli Proteins/physiology
• Gene Expression/genetics
• Gene Expression Regulation, Bacterial/physiology
• Genetic Variation/genetics
• Models, Genetic
• Plasmids/genetics
• Transfection/methods

• Anabaena/classification
• Anabaena/genetics
• Cloning, Molecular
• DNA Replication/genetics
• DNA Transposable Elements/genetics
• Escherichia coli Proteins/genetics
• Genetic Complementation Test
• Genetic Vectors/genetics
• Mutation
• Plasmids/genetics

• Antibodies, Bispecific/biosynthesis
• Antibodies, Bispecific/genetics
• Antibodies, Bispecific/immunology
• Biotechnology/methods
• CD3 Complex/genetics
• CD3 Complex/immunology
• Escherichia coli/genetics
• Female
• Gene Deletion
• Humans
• Immunotherapy/methods
• Ovarian Neoplasms/immunology
• Ovarian Neoplasms/therapy
• Plasmids/genetics
• Plasmids/immunology
• Rec A Recombinases/genetics
• Recombinant Proteins/biosynthesis
• Recombinant Proteins/genetics
• Recombinant Proteins/immunology

• Biofilms/growth & development
• Culture Media
• Escherichia coli/genetics
• Escherichia coli/growth & development
• Gene Transfer, Horizontal
• Plasmids/genetics
• Transformation, Bacterial/genetics

• Base Sequence
• Codon/genetics
• Codon, Terminator/genetics
• Escherichia coli/genetics
• Lac Operon/genetics
• Peptide Chain Termination, Translational/genetics
• Plasmids/genetics
• Protein Biosynthesis/genetics
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA, Transfer/genetics
• RNA, Transfer/metabolism
• RNA, Transfer, Amino Acyl/genetics
• RNA, Transfer, Amino Acyl/metabolism
• Ribosomes/genetics
• Ribosomes/metabolism
• beta-Galactosidase/metabolism

• Bacterial Adhesion
• Bacterial Proteins/metabolism
• Biofilms/growth & development
• Escherichia coli/physiology
• Locomotion
• Mutagenesis, Site-Directed
• Mutation
• Penicillin-Binding Proteins/genetics
• Penicillin-Binding Proteins/metabolism
• Plasmids/genetics
• Pseudomonas aeruginosa/physiology
• Sequence Deletion
• beta-Lactamases/metabolism

• R01 GM061019 NIGMS NIH HHS

• Animals
• Bacteriophage lambda/genetics
• Chickens
• DNA Damage
• DNA, Bacterial/genetics
• Deoxyribonuclease EcoRI/metabolism
• Escherichia coli/genetics
• Escherichia coli/metabolism
• Escherichia coli Proteins/genetics
• Escherichia coli Proteins/metabolism
• Exodeoxyribonucleases/genetics
• Exodeoxyribonucleases/metabolism
• Immunoglobulin Variable Region/biosynthesis
• Immunoglobulin Variable Region/genetics
• Peptide Library
• Recombination, Genetic
• beta-Lactamases/genetics