Biochemistry, Genetics and Molecular Biology › Genetics

Bacterial Genetics and Biotechnology

Works Count: 55162

Wikipedia

Description

This cluster of papers covers a wide range of topics related to bacterial physiology, genetics, and molecular mechanisms. It includes studies on gene regulation, antibiotic resistance, cellular homeostasis, signal transduction, membrane proteins, RNA regulation, iron metabolism, and microbial persistence.

Keywords

Bacterial Physiology; Gene Regulation; Antibiotic Resistance; Cellular Homeostasis; Signal Transduction; Bacterial Genetics; Membrane Proteins; RNA Regulation; Iron Metabolism; Microbial Persistence

A short course in bacterial genetics : a laboratory manual and handbook for Escherichia coli and related bacteria

1992-01-01

Jeffrey H Miller

A sequel to Experiments in Molecular Genetics (Cold Spring Harbor Lab. Press, 1972) for those doing genetic or recombinant DNA work with E. coli or similar organisms. The spiral-bound manual … A sequel to Experiments in Molecular Genetics (Cold Spring Harbor Lab. Press, 1972) for those doing genetic or recombinant DNA work with E. coli or similar organisms. The spiral-bound manual includes 34 detailed experiments with step-by-step protocols and clear diagrams that demonstrate major concep

Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria

1990-11-01

Vı́ctor de Lorenzo , Marta Herrero , Ute Jakubzik +1 more

A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into … A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.

View PDF Chat

Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria

1990-11-01

Marta Herrero , Vı́ctor de Lorenzo , Kenneth N. Timmis

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features … A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).

View PDF Chat

[6] Use of T7 RNA polymerase to direct expression of cloned genes

1990-01-01

F. William Studier , Alan H. Rosenberg , John J. Dunn +1 more

View PDF Chat

Pedigrees of some mutant strains of Escherichia coli K-12

1972-12-01

B Bachmann

View PDF Chat

The complete general secretory pathway in gram-negative bacteria

1993-03-01

A. P. Pugsley

The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of … The unifying feature of all proteins that are transported out of the cytoplasm of gram-negative bacteria by the general secretory pathway (GSP) is the presence of a long stretch of predominantly hydrophobic amino acids, the signal sequence. The interaction between signal sequence-bearing proteins and the cytoplasmic membrane may be a spontaneous event driven by the electrochemical energy potential across the cytoplasmic membrane, leading to membrane integration. The translocation of large, hydrophilic polypeptide segments to the periplasmic side of this membrane almost always requires at least six different proteins encoded by the sec genes and is dependent on both ATP hydrolysis and the electrochemical energy potential. Signal peptidases process precursors with a single, amino-terminal signal sequence, allowing them to be released into the periplasm, where they may remain or whence they may be inserted into the outer membrane. Selected proteins may also be transported across this membrane for assembly into cell surface appendages or for release into the extracellular medium. Many bacteria secrete a variety of structurally different proteins by a common pathway, referred to here as the main terminal branch of the GSP. This recently discovered branch pathway comprises at least 14 gene products. Other, simpler terminal branches of the GSP are also used by gram-negative bacteria to secrete a more limited range of extracellular proteins.

View PDF Chat

Protein phosphorylation and regulation of adaptive responses in bacteria

1989-12-01

Jeff Stock , Alexander J. Ninfa , Ann Stock

Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to … Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry.

View PDF Chat

FtsZ ring structure associated with division in Escherichia coli

1991-11-01

Erfei Bi , Joe Lutkenhaus

A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system

1981-01-01

Mark M. Garner , Arnold Revzin

The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in … The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified.

View PDF Chat

Elevated recombination rates in transcriptionally active DNA

1989-02-01

Barbara J. Thomas , Rodney Rothstein

One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.

1989-04-01

Cathie T. Chung , Sanna‐Mari Niemelä , R.H. Miller

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% … We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

View PDF Chat

Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

1980-04-01

M J Casadaban , Stanley N. Cohen

Interdependence of Cell Growth and Gene Expression: Origins and Consequences

2010-11-18

Matthew P. Scott , Carl W. Gunderson , Eduard M. Mateescu +2 more

In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic … In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells

1979-01-01

Manuel Dagert , S. Dusko Ehrlich

Construction of Biologically Functional Bacterial Plasmids In Vitro

1973-11-01

Stanley N. Cohen , Annie Chang , Herbert W. Boyer +1 more

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by … The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins.

View PDF Chat

Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant

1995-01-01

Peter Cherepanov , Wilfried Wackernagel

A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants

1998-05-01

Tung T. Hoang , RoxAnn R. Karkhoff-Schweizer , Alecksandr Kutchma +1 more

Molecular basis of base substitution hotspots in Escherichia coli

1978-08-01

Christine Coulondre , Jeffrey H Miller , Philip J. Farabaugh +1 more

The SOS regulatory system of Escherichia coli

1982-05-01

John W. Little , David W. Mount

Genetic regulatory mechanisms in the synthesis of proteins

1961-06-01

François Jacob , Jacques Monod

Carbon catabolite repression in bacteria: many ways to make the most out of nutrients

2008-07-16

Boris Görke , Jörg Stülke

Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes

1995-12-01

Michael E. Kovach , Philip H. Elzer , D. Steven Hill +4 more

Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.

1975-10-01

Michael Grunstein , David S. Hogness

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a … A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.

View PDF Chat

From the regulation of peptidoglycan synthesis to bacterial growth and morphology

2011-12-28

Athanasios Typas , Manuel Banzhaf , Carol A. Gross +1 more

View PDF Chat

Improved single and multicopy lac-based cloning vectors for protein and operon fusions

1987-01-01

Robert W. Simons , Fariba Houman , Nancy Kleckner

Vectors for selective expression of cloned DNAs by T7 RNA polymerase

1987-01-01

Alan H. Rosenberg , Barbara N. Lade , Chui Dao-shan +3 more

Making sense of it all: bacterial chemotaxis

2004-12-01

George H. Wadhams , Judith P. Armitage

A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.

1985-02-01

Stanley Tabor , C C Richardson

The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% … The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.

View PDF Chat

A colony bank containing synthetic CoI EI hybrid plasmids representative of the entire E. coli genome

1976-09-01

Louise Clarke , John Carbon

λ ZAP: a bacteriophage λ expression vector within vivoexcision properties

1988-01-01

Jay M. Short , Joseph M. Fernandez , Joseph A. Sorge +1 more

A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed.In E. coli a phagemid, pBluescnpt SK(-), contained within the vector, can be excised by f1 or M13 helper … A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed.In E. coli a phagemid, pBluescnpt SK(-), contained within the vector, can be excised by f1 or M13 helper phage.The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restrction digestion and ligation.This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the fI bacteriophage origin of replication (1).Six of 21 restriction sites in the excised pBluescript SK polyflinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP.Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZpromoter as fusion proteins.The features of this vector significantly increase the rate at which clones can be isolated and analyzed.The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes.Putative actin clones were excised from the lambda vector and identified by DNA sequencing.The ability of lambda ZAP to serve as a vector for the constnuction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.

View PDF Chat

Regulatory RNAs in Bacteria

2009-02-01

L. Waters , Gisela Storz

View PDF Chat

Structure and Mechanism of the Lactose Permease of Escherichia coli

2003-08-01

Jeff Abramson , И. Н. Смирнова , Vladimir N. Kasho +3 more

Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 … Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, β-D-galactopyranosyl-1-thio-β-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.

The Bacterial Cell Envelope

2010-04-14

Thomas J. Silhavy , Daniel Kahne , Scott S. Walker

Thomas J. Silhavy1, Daniel Kahne2 and Suzanne Walker2 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138; and … Thomas J. Silhavy1, Daniel Kahne2 and Suzanne Walker2 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138; and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Correspondence: tsilhavy{at}princeton.edu

View PDF Chat

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

2008-02-11

E. Sauvage , Frédéric Kerff , Mohammed Terrak +2 more

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such … Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

View PDF Chat

Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection

2006-01-01

Tomoya Baba , Takeshi Ara , Miki Hasegawa +7 more

View PDF Chat

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

2000-05-30

Kirill A. Datsenko , Barry L. Wanner

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, … We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage λ Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB , cyaA , lacZYA , ompR - envZ , phnR , pstB , pstCA , pstS , pstSCAB - phoU , recA , and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics

2007-09-01

Michael A. Kohanski , Daniel J. Dwyer , Boris Hayete +2 more

View PDF Chat

Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.

1982-01-01

Peter J. Southern , Paul Berg

A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are … A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

2003-12-01

Hiroshi Nikaido

SUMMARY Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria … SUMMARY Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

View PDF Chat

An efficient recombination system for chromosome engineering in Escherichia coli

2000-05-16

Daiguan Yu , Hilary Ellis , E-Chiang Lee +3 more

A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated … A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

View PDF Chat

Physiological and genetic responses of bacteria to osmotic stress

1989-03-01

László N. Csonka

The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary … The capacity of organisms to respond to fluctuations in their osmotic environments is an important physiological process that determines their abilities to thrive in a variety of habitats. The primary response of bacteria to exposure to a high osmotic environment is the accumulation of certain solutes, K+, glutamate, trehalose, proline, and glycinebetaine, at concentrations that are proportional to the osmolarity of the medium. The supposed function of these solutes is to maintain the osmolarity of the cytoplasm at a value greater than the osmolarity of the medium and thus provide turgor pressure within the cells. Accumulation of these metabolites is accomplished by de novo synthesis or by uptake from the medium. Production of proteins that mediate accumulation or uptake of these metabolites is under osmotic control. This review is an account of the processes that mediate adaptation of bacteria to changes in their osmotic environment.

View PDF Chat

Iron Metabolism in Pathogenic Bacteria

2000-10-01

Colin Ratledge , Lynn G. Dover

▪ Abstract The ability of pathogens to obtain iron from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host is central to whether they live or die. To combat … ▪ Abstract The ability of pathogens to obtain iron from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host is central to whether they live or die. To combat invading bacteria, animals go into an iron-withholding mode and also use a protein (Nramp1) to generate reactive oxygen species in an attempt to kill the pathogens. Some invading bacteria respond by producing specific iron chelators—siderophores—that remove the iron from the host sources. Other bacteria rely on direct contact with host iron proteins, either abstracting the iron at their surface or, as with heme, taking it up into the cytoplasm. The expression of a large number of genes (>40 in some cases) is directly controlled by the prevailing intracellular concentration of Fe(II) via its complexing to a regulatory protein (the Fur protein or equivalent). In this way, the biochemistry of the bacterial cell can accommodate the challenges from the host. Agents that interfere with bacterial iron metabolism may prove extremely valuable for chemotherapy of diseases.

Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements

1997-03-15

Rainer Lutz

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coliof several … Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coliof several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracy-cline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia colihost strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

View PDF Chat

Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum

1994-07-01

Andreas Schäfer , Andreas Tauch , Wolfgang Jäger +3 more

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter

1995-07-01

Luz‐Maria Guzman , Dominique Belin , M. Carson +1 more

We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this … We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

View PDF Chat

Essential Bacillus subtilis genes

2003-04-07

Kazuo Kobayashi , S. Dusko Ehrlich , Alberto Albertini +7 more

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, … To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among ≈4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden–Meyerhof–Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

View PDF Chat

On the Regulation of DNA Replication in Bacteria

1963-01-01

F Jacob , Sydney Brenner , François Cuzin

The deoxyribonucleotide sequence containing the genetic information of a cell participates in two distinct chemical processes. In the first one, generally called replication, free deoxyribonucleotides are linearly assembled by specific … The deoxyribonucleotide sequence containing the genetic information of a cell participates in two distinct chemical processes. In the first one, generally called replication, free deoxyribonucleotides are linearly assembled by specific base-pairing to form an identical sequence, or replica of the original structure. The second process, or transcription, allows the genetic material to perform its physiological functions consisting essentially in the production of specific proteins at a suitable rate. As a first step, transcription involves the production of messengers which carry to the protein-forming-centers the information necessary to specify the structure of the polypeptide chains. Messenger synthesis by DNA is a process probably closely similar to that of replication, with the differences that it involves ribo-, instead of deoxyribonucleotides and that, in all likelihood, only one of the DNA strands is used for copying into an RNA transcript.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

2006-12-01

Josef Deutscher , Christof Francke , Pieter W. Postma

SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To … SUMMARY The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

View PDF Chat

Lipid A Modification Systems in Gram-Negative Bacteria

2007-06-07

Christian R.H. Raetz , C. Michael Reynolds , M. Stephen Trent +1 more

The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the … The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines.

View PDF Chat

FACS-optimized mutants of the green fluorescent protein (GFP)

1996-01-01

Brendan P. Cormack , Raphael H. Valdivia , Stanley Falkow

We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) … We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

Direct and indirect pathways linking the Lon protease to motility behaviors in the pathogen Pseudomonas aeruginosa

2025-06-25

Aswathy Kallazhi , A.F.M.S. Rahman , Ute Römling +1 more | PLoS Pathogens

The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa , … The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa , lon loss-of-function mutants exhibit multiple phenotypic defects in motility, virulence, antibiotic tolerance and biofilm formation. However, only a couple of native substrate proteins of Lon are described in P. aeruginosa until now and most of the phenotypes associated with Lon remain unexplained. Here, we searched for novel Lon substrates in P. aeruginosa by analyzing proteome-wide changes in protein levels and stabilities following lon overexpression. Our search yielded a large number of putative Lon substrates with diverse cellular functions, including metabolic enzymes, stress proteins and a significant fraction of motility-related proteins. In vitro degradation assays confirmed the metabolic protein SpeH, the heat shock protein IbpA as well as seven proteins involved in flagella- and type IV pilus-mediated motility as novel substrates of Lon. The new motility-associated substrates include both key regulators of motility (FliA, RpoN, AmrZ) as well as structural flagellar components (FliG, FliS and FlgE). Further, by isolating suppressor mutations bypassing the motility defect of lon- cells, we reveal that Lon-dependent degradation of the specific substrate SulA, a cell division inhibitor, is crucial for ensuring proper cell division and motility under optimal conditions. In sum, our work highlights Lon’s regulatory role in degrading functional proteins involved in critical cellular processes and contributes to a better molecular understanding of the pathways underlying Pseudomonas pathogenicity.

Nonequilibrium polysome dynamics promote chromosome segregation and its coupling to cell growth in Escherichia coli

2025-06-24

Alexandros Papagiannakis , Qiwei Yu , Sander K. Govers +3 more | eLife

Chromosome segregation is essential for cellular proliferation. Unlike eukaryotes, bacteria lack cytoskeleton-based machinery to segregate their chromosomal DNA (nucleoid). The bacterial ParABS system segregates the duplicated chromosomal regions near the … Chromosome segregation is essential for cellular proliferation. Unlike eukaryotes, bacteria lack cytoskeleton-based machinery to segregate their chromosomal DNA (nucleoid). The bacterial ParABS system segregates the duplicated chromosomal regions near the origin of replication. However, this function does not explain how bacterial cells partition the rest (bulk) of the chromosomal material. Furthermore, some bacteria, including Escherichia coli , lack a ParABS system. Yet, E. coli faithfully segregates nucleoids across various growth rates. Here, we provide theoretical and experimental evidence that polysome production during chromosomal gene expression helps compact, split, segregate, and position nucleoids in E. coli through nonequilibrium dynamics that depend on polysome synthesis, degradation (through mRNA decay), and exclusion from the DNA meshwork. These dynamics inherently couple chromosome segregation to biomass growth across nutritional conditions. Halting chromosomal gene expression and thus polysome production immediately stops sister nucleoid migration, while ensuing polysome depletion gradually reverses nucleoid segregation. Redirecting gene expression away from the chromosome and toward plasmids causes ectopic polysome accumulations that are sufficient to drive aberrant nucleoid dynamics. Cell width enlargement experiments suggest that limiting the exchange of polysomes across DNA-free regions ensures nucleoid segregation along the cell length. Our findings suggest a self-organizing mechanism for coupling nucleoid compaction and segregation to cell growth without the apparent requirement of regulatory molecules.

Dynamic plasmid clustering in Bacteria: Influence of transcriptional activity and host cellular states

2025-06-23

Guanlin Wang , Yi-Ren Chang | Biochemistry and Biophysics Reports

A promiscuous Bcd amino acid dehydrogenase promotes biofilm development in Bacillus subtilis

2025-06-21

David Ranava , Stephen M. Lander , S.M. Kuan +3 more | npj Biofilms and Microbiomes

Glutamate dehydrogenase (GDH) resides at the crossroads of nitrogen and carbon metabolism, catalyzing the reversible conversion of L-glutamate to α-ketoglutarate and ammonium. GDH paralogs are ubiquitous across most species, presumably … Glutamate dehydrogenase (GDH) resides at the crossroads of nitrogen and carbon metabolism, catalyzing the reversible conversion of L-glutamate to α-ketoglutarate and ammonium. GDH paralogs are ubiquitous across most species, presumably enabling functional specialization and genetic compensation in response to diverse conditions. Staphylococcus aureus harbors a single housekeeping GDH (GudB), whereas Bacillus subtilis encodes both a major and a minor GDH, GudB and RocG, respectively. In an unsuccessful attempt to identify an alternative GDH in S. aureus, we serendipitously discovered previously unrecognized GDH activity in two metabolic enzymes of B. subtilis. The hexameric Val/Leu/Ile dehydrogenase Bcd (formerly YqiT) catabolizes branched-chain amino acids and to a lesser extent glutamate using NAD+ as a cofactor. Removal of gudB and rocG unmasks the dual NAD(P)+-dependent GDH activity of RocA, which otherwise functions as a 3-hydroxy-1-pyrroline-5-carboxylate dehydrogenase. Bcd homologs are prevalent in free-living and obligate bacteria but are absent in most, if not all, staphylococci. Despite low sequence homology, Bcd structurally resembles the GudB/RocG family and can functionally compensate for the loss of GudB in S. aureus. Bcd is essential for the full maturation of biofilms. B. subtilis lacking GDHs exhibits severe impairments in rugose architecture and colony expansion of biofilms. This study underscores the importance of metabolic redundancy and highlights the critical role of substrate promiscuity in GDHs during biofilm development.

Inhibiting Lipopolysaccharide Biogenesis: The More You Know the Further You Go

2025-06-20

Caitlin B. Moffatt , Bailey A. Plaman , Sebastian J. Rowe +4 more | Annual Review of Biochemistry

Gram-negative bacteria are intrinsically resistant to many antibiotics because they are surrounded by an outer membrane that creates a robust permeability barrier. The outer membrane has an unusual asymmetric structure … Gram-negative bacteria are intrinsically resistant to many antibiotics because they are surrounded by an outer membrane that creates a robust permeability barrier. The outer membrane has an unusual asymmetric structure with a periplasmic leaflet composed of phospholipids and an outer leaflet composed of lipopolysaccharides. Because lipid biosynthesis is completed in the inner membrane of these didermic bacteria, these components must be transported across the cell envelope and properly assembled to expand the outer membrane during growth and division. Lipopolysaccharide molecules are transported over a multi-protein transenvelope bridge that is powered by ATP hydrolysis in the cytoplasm. This review discusses how this bridge is assembled and functions and how lipopolysaccharide transport is regulated to ensure balanced growth of all envelope layers. A combination of approaches and new experimental tools have significantly advanced our understanding of this molecular machine and contributed to the development of new antimicrobials that interfere with transport.

Iterativein vivocut‘n’paste of chromosomal loci inEscherichia coliK-12 using synthetic DNA

2025-06-19

Sebastian Gude , Andreas Birk Bertelsen , Morten H. H. Nørholm | bioRxiv (Cold Spring Harbor Laboratory)

Abstract We have developed a simple and highly efficient in vivo method to iteratively relocate functional chromosomal loci onto an episome in Escherichia coli by utilizing synthetic DNA fragments. In … Abstract We have developed a simple and highly efficient in vivo method to iteratively relocate functional chromosomal loci onto an episome in Escherichia coli by utilizing synthetic DNA fragments. In this in vivo cut’n’paste procedure, “cutting” is executed by the RNA-guided DNA endonuclease Cas9 and a set of guides, while “pasting” is facilitated by the phage λ Red recombinase, which are all synthesized on easily curable plasmids. To demonstrate the utility of this approach, we commercially obtained synthetic DNA fragments containing locus-specific homology regions, antibiotic marker cassettes, and standardized Cas9 target sequences. By using these locus-specific synthetic DNA fragments, we relocated 7 functional chromosomal loci. Scarless relocation mutants of the trg , aer , tsr , malHM , malQT , macB - nadA , and folA chromosomal loci were obtained as antibiotic-resistant isolates by combining Cas9 counterselection with the restoration of an antibiotic marker cassette. The additional antibiotic marker cassettes and standardized Cas9 target sequences present in the synthetic DNA fragments are inherently eliminated upon completion of the procedure, enabling iterative processing of loci. This procedure should be widely useful, especially in genome (re-)engineering of E. coli and other bacteria because the procedure can be done on wild-type cells.

Regulatory Interplay Between FtsH, HflKC and YidC in Bacterial Membrane Protein Biogenesis and Quality Control

2025-06-18

Mehmet Çalıseki , Sarah Zorman , Christiane Schaffitzel +1 more | Research Square (Research Square)

<title>Abstract</title> The quality control of membrane proteins is essential for maintaining cellular homeostasis, as misfolded or damaged proteins can disrupt essential cellular functions. FtsH, a membrane-bound AAA + metalloprotease, is … <title>Abstract</title> The quality control of membrane proteins is essential for maintaining cellular homeostasis, as misfolded or damaged proteins can disrupt essential cellular functions. FtsH, a membrane-bound AAA + metalloprotease, is central to bacterial proteostasis, responsible for degrading misfolded or damaged membrane proteins. YidC, a membrane protein insertase, facilitates the folding, insertion, and assembly of membrane proteins into the lipid bilayer. This study investigates the physical interaction between FtsH, HflKC, and YidC, indicating a potential functional relationship between these proteins in maintaining membrane protein quality control in bacteria. Overexpression of YidC in <italic>Escherichia coli</italic> led to a disruption in the interaction between FtsH and its regulatory proteins HflK and HflC, possibly due to competition for binding sites. This was supported by the depletion of HflK and HflC in both Western blot and mass spectrometry analyses using detergent-solubilized membrane extracts. Additionally, the co-overexpression of FtsH and YidC induced cellular stress, as evidenced by the increased recruitment of stress-related proteins such as GroEL and DnaK. These findings suggest that FtsH and YidC collaborate in membrane protein biogenesis and participate in stress-responsive regulatory mechanisms that contribute to protein homeostasis.

Gene age and genome organization in Escherichia coli and Bacillus subtilis

2025-06-18

Carsten Jers , Hrvoje Mišetić , Vaishnavi Ravikumar +4 more | Frontiers in Microbiology

Using genomic phylostratigraphy, we examined the organization of Escherichia coli and Bacillus subtilis genomes from the perspective of evolutionary age of their genes. Phylostratigraphy analysis classifies individual genes into age-related … Using genomic phylostratigraphy, we examined the organization of Escherichia coli and Bacillus subtilis genomes from the perspective of evolutionary age of their genes. Phylostratigraphy analysis classifies individual genes into age-related bins, called phylostrata. Based on this analysis, several common features emerged in the genomes of the two model bacteria. More recent genes tend to be shorter and are expressed less frequently, or only in specific conditions. In terms of genomic location, new genes are enriched in areas containing prophages, suggesting a link with horizontal gene transfer. Interestingly, while most bacterial transcription regulators belong to the oldest phylostrata, they regulate expression of both older and more recent genes alike. A large fraction of bacterial operons contains genes from different phylostrata. This suggests that newer genes are integrated in the existing framework for regulating gene expression, and that the establishment of new regulatory circuits typically do not accompany acquisition of new genes. One striking difference between E. coli and B. subtilis genomes was observed. About 87.0% of all E. coli genes belong to the evolutionary oldest physlostratum. In B. subtilis , this number is only 71.8%, indicating a more eventful evolutionary past in terms of acquisition of new genes, either by gene emergence or by horizontal transfer.

Cyclic-di-AMP modulates cellular turgor in response to defects in bacterial cell wall synthesis

2025-06-17

Anna P. Brogan , Paola Bardetti , Enrique Rojas +1 more | Nature Microbiology

Bacteria deliver a microtubule-binding protein into mammalian cells to promote colonization

2025-06-17

Michael S. Costello , Bryan C. Neumann , Bonnie J. Cuthbert +7 more | bioRxiv (Cold Spring Harbor Laboratory)

Abstract Pathogenic Bordetella bacteria infect the ciliated respiratory epithelia of mammalian and avian hosts. Several bacterial proteins mediate host cell adhesion, but filamentous hemagglutinin (FhaB) is a principal adhesin because … Abstract Pathogenic Bordetella bacteria infect the ciliated respiratory epithelia of mammalian and avian hosts. Several bacterial proteins mediate host cell adhesion, but filamentous hemagglutinin (FhaB) is a principal adhesin because mutants lacking this protein exhibit profound colonization defects. Here, we show that FhaB carries a C-terminal microtubule-binding domain (FhaB-CT), which is translocated into the host-cell cytoplasm to promote bacterial colonization. Cryogenic electron microscopy of microtubule-bound FhaB-CT shows that the domain binds primarily to α-tubulin through a network of polar interactions. Live-cell microscopy of infected tracheal explants reveals that FhaB-CT delivery is required for Bordetella to occupy a niche at the base of cilia on airway epithelia. Finally, we demonstrate that the microtubule-binding domain is required for long-term colonization of the mouse nasal cavity by B. pertussis . These observations suggest that the FhaB-CT domain is delivered into motile cilia, where it interacts with axonemal microtubules. We propose that Bordetella initially adhere to the tips of cilia, then deploy multiple FhaB adhesin molecules to migrate to the base of the cilial forest. This mechanism enables Bordetella to resist removal by the mucociliary ‘escalator’ that clears the respiratory tract of microbes and debris.

Tuning the interaction of a ParA-type ATPase with its partner separates bacterial organelle positioning from partitioning

2025-06-17

Anthony G. Vecchiarelli , Jordan Byrne , Hema M. Swasthi +3 more | Research Square (Research Square)

<title>Abstract</title> The maintenance of carboxysome distribution (Mcd) system comprises the proteins McdA and McdB, which spatially organize carboxysomes to promote efficient carbon fixation and ensure their equal inheritance during cell … <title>Abstract</title> The maintenance of carboxysome distribution (Mcd) system comprises the proteins McdA and McdB, which spatially organize carboxysomes to promote efficient carbon fixation and ensure their equal inheritance during cell division. McdA, a member of the ParA/MinD family of ATPases, forms dynamic gradients on the nucleoid that position McdB-bound carboxysomes. McdB belongs to a widespread but poorly characterized class of ParA/MinD partner proteins, and the molecular basis of its interaction with McdA remains unclear. Here, we demonstrate that the N-terminal 20 residues of <italic>H. neapolitanus</italic> McdB are both necessary and sufficient for interaction with McdA. Within this region, we identify three lysine residues whose individual substitution modulates McdA binding and leads to distinct carboxysome organization phenotypes. Notably, lysine 7 (K7) is critical for McdA interaction: substitutions at this site result in the formation of a single carboxysome aggregate positioned at mid-nucleoid. This phenotype contrasts with that of an McdB deletion, in which carboxysome aggregates lose their nucleoid association and become sequestered at the cell poles. These findings suggest that weakened McdA–McdB interactions are sufficient to maintain carboxysome aggregates on the nucleoid but inadequate for partitioning individual carboxysomes across it. We propose that, within the ParA/MinD family of ATPases, cargo positioning and partitioning are mechanistically separable: weak interactions with the cognate partner can mediate positioning, whereas effective partitioning requires stronger interactions capable of overcoming cargo self-association forces.

Simple and robust in vivo engineering of plasmid DNA at any copy number in Escherichia coli

2025-06-17

Ana Gabriela Veiga Sepulchro , Hendrikje C J Kozlowski , Morten H. H. Nørholm | Communications Biology

Plasmids and the model bacterium Escherichia coli are at the heart of recombinant gene technologies. Plasmids are handled in test tubes with enzymes such as restriction endonucleases, ligases, and polymerases. … Plasmids and the model bacterium Escherichia coli are at the heart of recombinant gene technologies. Plasmids are handled in test tubes with enzymes such as restriction endonucleases, ligases, and polymerases. However, with the increasing demand for larger and more complex designs, in vitro manipulation constitutes a bottleneck. By combining recombination with genetic selection, in vivo manipulation of genomic DNA is becoming routine but is yet to be developed as a versatile and reliable way to make plasmid DNA. Here, we present a robust methodology for plasmid recombineering in E. coli using a triple-selection system customized for efficient performance at any copy number. Equipped with this genetic selection cassette, we generate a toolbox of plasmids in a standardized framework with popular genetic modules. By reducing the time and resources for making recombinant DNA, this approach should enable automation and accelerate the development of biological solutions.

CRISPRi screening reveals E. coli ’s anaerobic-like respiratory adaptations to gentamicin: membrane depolarization by CpxR

2025-06-16

Donghui Choe , Eun‐Ju Lee , Yoseb Song +5 more | mSystems

ABSTRACT Bacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in … ABSTRACT Bacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in Escherichia coli exposed to gentamicin, aiming to understand the cellular defense mechanisms. Our findings revealed that ribosomal proteins, ribosome-associated proteins, toxin-antitoxin systems, and outer membrane proteins strongly influence the fitness of E. coli in gentamicin. Notably, gentamicin-induced fitness changes resembled those under anaerobic conditions, where resistance to gentamicin was observed. Specifically, genes related to the biosynthesis of cofactors and electron carriers, crucial for the respiratory system, showed reduced essentiality under both gentamicin and anaerobic conditions, suggesting a disruption in membrane potential leading to limited gentamicin uptake. Transcriptomic and genome-wide binding analyses identified the two-component system CpxR as a key regulator of respiratory systems in response to gentamicin. Our study provides insights into cellular defense mechanisms, offering potential strategies for combating antibiotic resistance. IMPORTANCE Bacteria can adapt to a variety of stressful environments, including antibiotic exposure. The mechanisms underlying antibiotic resistance remain an active area of investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) interference enables specific silencing of gene expression, allowing researchers to assess the fitness effects of gene knockdowns under given conditions. Using genome-wide CRISPR interference screening on Escherichia coli exposed to gentamicin, we identified anaerobic-like fitness effects of genes involved in respiration and the maintenance of membrane potential—key processes that facilitate gentamicin entrance into the cell. Transcriptomic analysis and immunoprecipitation assays further indicated that the two-component system CpxR modulates respiratory adaptations in response to gentamicin challenge. These findings shed light on the development of antibiotic resistance in bacteria and may offer new insight into strategies for treating gentamicin-resistant pathogens.

Knockout of tusA facilitates flagella formation and cationic antimicrobial resistance by disrupting Fur transcriptional regulation in Escherichia coli

2025-06-16

Kazuya Ishikawa , Koso NAKATA , Kazuyuki Furuta +1 more | bioRxiv (Cold Spring Harbor Laboratory)

tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Recent studies have reported that tusA deficiency affects iron-sulfur (Fe-S) homeostasis and cluster … tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Recent studies have reported that tusA deficiency affects iron-sulfur (Fe-S) homeostasis and cluster formation in Escherichia coli; however, its association with this phenotype remains unclear. In this study, we analyzed the phenotype of tusA-deficient E. coli (?tusA) and its underlying mechanisms using RNA sequencing. We observed that tusA deletion disrupted the expression of genes regulated by the ferric uptake regulator Fur or Fur-regulated transcription factors (flagellar transcriptional regulators D and C [FlhDC] and fumarate and nitrate reduction regulator [Fnr]). Increased expression of flhDC, which is the master regulator of flagellar genes facilitated flagella formation even under conditions in which the wild-type formed few flagella. Additionally, ?tusA was resistant to cationic antibacterial agents, such as cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. This resistance is associated with the increased expression of ompX or ompF, regulated by Fur and Fnr, respectively. Notably, both enhanced flagella formation and resistance to cationic antibacterial agents caused by tusA deletion were abolished in the fur-deficient background. These findings indicate that impaired expression of the fur regulon, possibly because of impaired Fe-S cluster formation, induces multiple phenotypic alterations in ?tusA.

A novel AAA+ ATPase required for sporulation and stress response in Bacillus anthracis

2025-06-16

Nitika Sangwan , Ankur Bothra , Andrei P. Pomerantsev +7 more | bioRxiv (Cold Spring Harbor Laboratory)

AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel … AAA+ proteins function as molecular machines that utilize ATP to perform diverse cellular functions, including protein homeostasis, stress regulation, and cell cycle/developmental processes. In this study, we identified a novel AAA+ ATPase BAS PrkA in B. anthracis Sterne 34F2 which has 88 % protein homology to Bacillus subtilis PrkA. Conserved domain analysis confirms BAS PrkA has an N-terminal AAA+ ATPase domain with characteristic Walker A and Walker B motifs and a conserved secondary region of homology (SRH) domain, along with a C-terminal cAMP-dependent protein kinase domain. Based on Alpha Fold3 predicted structure, we classified BAS PrkA as part of Clade III of the AAA+ superfamily. Contrary to the reported enzymatic activity in B. subtilis PrkA, we observed that BAS PrkA has negligible protease and kinase activity under in-vitro conditions. Nonetheless, BAS PrkA plays a significant role in regulating sporulation. It is temporally expressed during Stages II to VI during sporulation. A null mutant of BAS PrkA exhibits severe sporulation defects, with reduced spore viability, and down regulation of genes related to spore-coat formation. These phenotypes were restored in a complementation strain expressing BAS PrkA ectopically. Additionally, the null mutant strain showed compromised growth under ionic-osmotic stress conditions. Analysis of the BAS PrkA interactome revealed enrichment of two proteins, ProA and EzrA, that are implicated in osmotic stress response and the sporulation process, respectively. These findings show that BAS PrkA plays a critical role in sporulation and osmotic stress response in B. anthracis .

A molecular conveyor belt-associated protein controls the rotational direction of the bacterial type 9 secretion system

2025-06-13

Abhishek Trivedi , Jacob Miratsky , Emma C. Henderson +2 more | mBio

ABSTRACT Many bacteria utilize the type 9 secretion system (T9SS) for gliding motility, surface colonization, and pathogenesis. This dual-function motor supports both gliding motility and protein secretion, where rotation of … ABSTRACT Many bacteria utilize the type 9 secretion system (T9SS) for gliding motility, surface colonization, and pathogenesis. This dual-function motor supports both gliding motility and protein secretion, where rotation of the T9SS plays a central role. Fueled by the energy of the stored proton motive force and transmitted through the torque of membrane-anchored stator units, the rotary T9SS propels an adhesin-coated conveyor belt along the bacterial outer membrane like a molecular snowmobile, thereby enabling gliding motion. However, the mechanisms controlling the rotational direction and gliding motility of T9SS remain elusive. Shedding light on this mechanism, we find that in the gliding bacterium Flavobacterium johnsoniae , deletion of the C-terminus of the conveyor belt-associated protein GldJ controls and, in fact, reverses the rotational direction of T9SS from counterclockwise (CCW) to clockwise (CW). This suggests that the interface between the conveyor belt-associated protein GldJ and the T9SS ring protein GldK plays an important role in controlling the directionality of T9SS, potentially by modulating its interaction with the stator complex GldLM, which drives motor rotation. Combined with MD simulation of the T9SS stator units GldLM, we suggest a “tri-component gearset” model where GldJ controls the rotational direction of its driver, the T9SS, thus providing adaptive sensory feedback to influence the motility of the gliding bacterium. IMPORTANCE The type 9 secretion system (T9SS) is fundamental to bacterial gliding motility, pathogenesis, and surface colonization. Our findings reveal that the C-terminal region of the conveyor belt-associated protein GldJ functions as a molecular switch which is capable of reversing the rotational direction of T9SS. Through the coordinated actions of the T9SS stator units (akin to a driving motor), the GldK ring (the gear that converts rotational energy into linear movement), and GldJ, this machinery forms a smart conveyor belt system reminiscent of flexible or cognitive mechanical conveyors. Such advanced conveyors can alter their direction to adapt to shifting demands. Here, we show that the bacterial T9SS similarly adjusts its rotational bias based on feedback from the conveyor belt-associated protein GldJ. This dual-role feedback mechanism underscores an evolved, controllable biological snowmobile, offering new avenues for studying how bacteria fine-tune motility in dynamic environments.

Analysis of 3'-seq data from multiple E. coli studies identifies diverging results sets and raw data characteristics despite similar collection conditions

2025-06-12

Quinlan Furumo , Michelle M. Meyer | bioRxiv (Cold Spring Harbor Laboratory)

Abstract 3-prime end sequencing (3’-seq) is a high-throughput sequencing technique that is used to specifically quantify the changes in 3’-end formation of transcripts in bacterial cells, which is increasingly being … Abstract 3-prime end sequencing (3’-seq) is a high-throughput sequencing technique that is used to specifically quantify the changes in 3’-end formation of transcripts in bacterial cells, which is increasingly being utilized to address fundamental questions regarding transcription termination and pausing across a range of different bacterial species. However, the growing number of 3’-seq studies is accompanied by an increase in study-specific 3’-seq data analysis approaches. Thus, differences in a number of factors including: experimental design, data collection approaches, analysis methodologies, and interpretation decisions, make it challenging to confidently compare results derived from different studies, even those that were performed on the same organism. To assess the potential severity of these discrepancies, we used PIPETS, a statistically robust and genome-annotation agnostic 3’-seq analysis package, to study Escherichia coli 3’-seq data sets from three different groups collected under similar conditions. By using a consistent analysis and results interpretation approach, we identified large disparities in the characteristics of the raw 3’-seq data between each of the studies, despite all three studies using the same strain and very similar reported experimental conditions. Additionally, we found strand-specific inconsistencies, with some data sets having reference strand 3’-seq read coverage distributions that differed greatly from the complement strand within the same replicate. Finally, when the 3’-seq distribution profiles of the three E. coli studies are compared to studies from four additional bacteria, we identified 3’-seq results clustering patterns that are not explained by phylogenetic similarity between organisms. With the large differences seen between data sets from the same organism as well as the inconsistencies seen between replicates from the same data sets, we urge the field to reconsider the assumptions around 3’-seq data homogeneity and move towards consistent analysis approaches, and cautious interpretation of the data. Graphical Abstract

A general mechanism for initiating the bacterial general stress response

2025-06-06

Rishika Baral , Laurent Blanchoin , Ramasamy P. Kumar +6 more | eLife

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates … The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in Bacillus subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. This coiled-coil linker transduction mechanism additionally suggests a resolution to the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Assembly and the gating mechanism of the Pel exopolysaccharide export complex PelBC of Pseudomonas aeruginosa

2025-06-05

Marius Benedens , Cristian Rosales , Sabine Straathof +5 more | Nature Communications

Abstract The pathogen Pseudomonas aeruginosa enhances its virulence and antibiotic resistance upon formation of durable biofilms. The exopolysaccharides Pel, Psl and alginate essentially contribute to the biofilm matrix, but their … Abstract The pathogen Pseudomonas aeruginosa enhances its virulence and antibiotic resistance upon formation of durable biofilms. The exopolysaccharides Pel, Psl and alginate essentially contribute to the biofilm matrix, but their secretion mechanisms are barely understood. Here, we reveal the architecture of the outer membrane complex PelBC for Pel export, where the essential periplasmic ring of twelve lipoproteins PelC is mounted on top of the nanodisc-embedded β-barrel PelB. The PelC assembly is stabilized by electrostatic contacts with the periplasmic rim of PelB and via the membrane-anchored acyl chains. The negatively charged interior of the PelB β-barrel forms a route for the cationic Pel exopolysaccharide. The β-barrel is sealed at the extracellular side, but molecular dynamic simulations suggest that the short loop Plug-S is sufficiently flexible to open a tunnel for the exopolysaccharide transport. This gating model is corroborated by single-channel conductivity measurements, where a deletion of Plug-S renders a constitutively open β-barrel. Our structural and functional analysis offers a comprehensive view on this pathogenicity-relevant complex and suggests the route taken by the exopolysaccharide at the final secretion step.

The outer membrane transporter FadL was involved in the uptake of C18 n-alkane for enhancement of biodegradation in Pseudomonas aeruginosa TJM4

2025-06-02

Qingyan Rao , Yong-Fang Li , Meijia Sun +3 more | Journal of Applied Microbiology

Abstract Aims The removal of n-alkanes by bacteria is a promising strategy for bioremediation. Especially, the transmembrane transport of n-alkanes is a critical intermediate process for transfer and adsorption. Herein, … Abstract Aims The removal of n-alkanes by bacteria is a promising strategy for bioremediation. Especially, the transmembrane transport of n-alkanes is a critical intermediate process for transfer and adsorption. Herein, the structure and function of two FadL outer membrane transporters in Pseudomonas aeruginosa TJM4 were explored to elucidate the impact on efficient removal of C18 n-alkanes. Methods and Results Phylogenetic analysis revealed considerable distinction in FadL sequences among strains involved in alkane catabolism. RT-qPCR analysis revealed that the expression of the two outer membrane transporter-encoding genes in strain TJM4, fadL1 and fadL2, was significantly upregulated in response to C18 n-alkane induction. To further investigate whether FadL was involved in the uptake and transport of C18 n-alkane, the fadL1 and fadL2 genes were deleted to generate mutants. Growth and degradation assays demonstrated that loss of the fadL gene reduced the ability of strain TJM4 to utilize and degrade C18 n-alkane, and FadL1 played a key role in the removal of C18 n-alkane. Moreover, the cell surface hydrophobicity of the mutants was significantly lower than that of the wild-type (WT) strain TJM4. However, the complementation of fadL gene restored alkane degradation capacity and CSH of those mutant strains. These findings supported the role of FadL in enhancement of n-alkane catabolism. Finally, molecular docking analysis revealed hydrophobic interactions between the two FadL transporters and C18 n-alkane, in which Val, Leu, Ile, and Ala played a role in both complexes. Conclusions Those results indicated that FadL was a pivotal step for removal of C18 n-alkane in strain TJM4. The pronounced differences in sequence between the two FadL transporters and their interaction sites with C18 n-alkane suggested that they might be substrate specific involved in the n-alkane transport pathway of strain TJM4.

The Escherichia coli RelB antitoxin C-terminus is essential for RelE toxin suppression and transcriptional repression

2025-06-01

Julia Tanquary , Ian Pavelich , Marcin Grabowicz +1 more | Journal of Biological Chemistry

A global perspective on autoinducer-2-mediated cell communication in prokaryotes

2025-06-01

Xiaozhen Liu , Zhiyan Wei , Mingming Yang +7 more | iScience

Lipid regulation of adenylyl cyclase Rv1625c from Mycobacterium tuberculosis by its membrane‐domain receptor

2025-06-01

Anita Schultz , Marius Landau , Andrei N. Lupas +1 more | FEBS Journal

The regulation of mammalian adenylyl cyclases by G‐protein‐coupled receptors and the Gsα subunit of trimeric G‐proteins has been extensively studied, whereas little is known about the regulation of their closely … The regulation of mammalian adenylyl cyclases by G‐protein‐coupled receptors and the Gsα subunit of trimeric G‐proteins has been extensively studied, whereas little is known about the regulation of their closely related bacterial cyclases. Here, we focused on the regulation of the adenylyl cyclase Rv1625c from Mycobacterium tuberculosis H37Rv . Rv1625c is a progenitor of mammalian congeners. Exclusively C 18 ‐mono‐unsaturated fatty acids, such as the cis‐ and trans‐isoforms of oleic and vaccenic acids, inhibited the Rv1625c holoenzyme with IC 50 concentrations around 10 μ m . The saturated C 18 fatty acid stearic acid was inactive. A soluble Rv1625c construct, which lacked the membrane domain, was not affected by the mono‐unsaturated C 18 fatty acids, i.e., the inhibition required the presence of the membrane domain, indicating a receptor–ligand interaction. Surprisingly, fatty acid inhibition of Rv1625c was strictly dependent on magnesium ions (Mg 2+ ) as a divalent cation for the substrate adenosine triphosphate (ATP). Although manganese ion (Mn 2+ )–ATP as a substrate greatly increased enzyme activity, Mn 2+ appeared to block intramolecular signal transduction from the membranous receptor domain to the catalytic effector domain. In summary, the results bolster the proposal that adenylyl cyclase regulation by fatty acids is an evolutionarily conserved signaling mode present in bacteria as well as in mammals.

FimC binds to the promoter region of agn43 to modulate autoaggregation

2025-05-30

Zhihao Wang , Xiangpeng Niu , Ningyuan Zhong +7 more | Frontiers in Cellular and Infection Microbiology

Avian pathogenic Escherichia coli (APEC) infection causes high mortality in chicks and leads to significant economic losses in the poultry industry. During the initial infection, APEC colonizes host cells using … Avian pathogenic Escherichia coli (APEC) infection causes high mortality in chicks and leads to significant economic losses in the poultry industry. During the initial infection, APEC colonizes host cells using type 1 fimbriae and subsequently forms biofilms, resulting in persistent and chronic infections. fimC is a chaperone protein associated with type 1 fimbriae and plays a crucial role in the assembly of fimbriae. However, its regulatory role in agn43-mediated autoaggregation remains unclear. By constructing fimC gene mutant strains, the autoaggregation, motility, biofilm formation, and the adhesion and invasion ability to HD-11 cells were examined. The transcriptome and the electrophoretic mobility shift assay (EMSA) were used to screen and verify the regulation of fimC on downstream genes. The results demonstrated that the lack of fimC, but not fimbriae, significantly increased autoaggregation (p < 0.001) while promoting the transcription of agn43 (p < 0.01). Transcriptomic analysis showed that the deletion of fimC caused significant changes in the gene transcription levels in a variety of pathways, such as flagellar synthesis, biofilm formation, quorum sensing, and bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) metabolism. Further investigation revealed that fimC directly interacted with the promoter region of agn43 and inhibited its transcription. In addition, both fimC and agn43 had regulatory effects on biofilm formation, motility, adhesion, and invasion. This study demonstrated that fimC acts as an atypical DNA-binding protein to regulate the transcription of agn43. It also highlights the importance of fimC in the biofilm formation and adhesion ability of APEC, which provides new insights into the functions of the fimbrial chaperone protein FimC.

Transcriptomic Insights into SlyA-Mediated Regulation of Motility and Secretion in Escherichia coli

2025-05-30

Sara Mohan , Prasanna Kumar Selvam , Karthick Vasudevan | Research Square (Research Square)

<title>Abstract</title> <italic>SlyA</italic>, a member of the MarR family of transcriptional regulators, has been implicated in regulating virulence, stress response, and motility in various Enterobacteriaceae. In this study, we performed transcriptomic … <title>Abstract</title> <italic>SlyA</italic>, a member of the MarR family of transcriptional regulators, has been implicated in regulating virulence, stress response, and motility in various Enterobacteriaceae. In this study, we performed transcriptomic profiling of <italic>Escherichia coli</italic> BW25113 wild-type and its isogenic <italic>SlyA</italic> knockout strain using publicly available RNA-Seq data, each consisting of four biological replicates. Differential gene expression analysis identified 925 significantly altered genes, including key components of the flagellar assembly pathway, such as <italic>fliA</italic>, <italic>flhB</italic>, and <italic>flgM</italic>. Gene Ontology and enrichment analyses revealed significant modulation of biological processes, including bacterial motility, chemotaxis, and protein secretion. Network analysis further highlighted a subset of hub genes involved in motility and secretion, suggesting their centrality in <italic>SlyA</italic>-mediated regulation. While the results indicate <italic>SlyA</italic>'s influence on multiple cellular pathways, these observations may reflect both direct and indirect regulatory effects. This transcriptomic analysis provides a computational foundation for future mechanistic studies and highlights pathways that may be targeted for antimicrobial development.

Molecular Insights into CLD Domain Dynamics and Toxin Recruitment of the HlyA E. coli T1SS

2025-05-30

Rocco Gentile , Stephan Schott‐Verdugo , Sakshi Khosa +5 more | bioRxiv (Cold Spring Harbor Laboratory)

Escherichia coli is a Gram-negative opportunistic pathogen causing nosocomial infections through the production of various virulence factors. Type 1 secretion systems (T1SS) contribute to virulence by mediating one-step secretion of … Escherichia coli is a Gram-negative opportunistic pathogen causing nosocomial infections through the production of various virulence factors. Type 1 secretion systems (T1SS) contribute to virulence by mediating one-step secretion of unfolded substrates directly into the extracellular space, bypassing the periplasm. A well-studied example is the hemolysin A (HlyA) system, which secretes the HlyA toxin in an unfolded state across the inner and outer membranes. T1SS typically comprise a homodimeric ABC transporter (HlyB), a membrane fusion protein (HlyD), and the outer membrane protein TolC. Some ABC transporters in T1SS also contain N-terminal C39 peptidase or peptidase-like (CLD) domains implicated in substrate interaction. Recent cryo-EM studies have resolved the inner-membrane complex as a trimer of HlyB homodimers with associated HlyD protomers. However, a full structural model including TolC remains unavailable. We present the first complete structural model of the HlyA T1SS, constructed using template- and MSA-based information and validated by SAXS. Molecular dynamics simulations provide insights into the function of the CLD domains, which are partially absent from existing cryo-EM structures. These domains may modulate transport by stabilizing specific conformations of the complex. Simulations with a C-terminal fragment of HlyA indicate that toxin binding occurs in the occluded conformation of HlyB, potentially initiating substrate transport through a single HlyB protomer before transitioning to an inward-facing state. HlyA binding also induces allosteric effects on HlyD, altering key residues involved in TolC recruitment. These results indicate how substrate recognition and transport are coupled and may support the development of antimicrobial strategies targeting the T1SS.

Characterization of the NRPS operon homolog for surfactin A and surfactin C synthesis in Bacillus spp.

2025-05-29

Koichi Ito , T. Adachi , Minenosuke Matsutani +4 more | Archives of Microbiology

Abstract Surfactin is a cyclic lipopeptide produced by Bacillus spp., consisting of a β-hydroxy fatty acid and a heptapeptide synthesized by non-ribosomal peptide synthetases. Surfactin congeners (A, B, and C) … Abstract Surfactin is a cyclic lipopeptide produced by Bacillus spp., consisting of a β-hydroxy fatty acid and a heptapeptide synthesized by non-ribosomal peptide synthetases. Surfactin congeners (A, B, and C) differ in amino acid substitutions, with Leu7 in surfactin A replaced by Val in B and Ile in C. Our LC–MS analysis revealed that the elution profiles of surfactin-producing strains could be classified into two distinct patterns under identical culture conditions, corresponding to surfactin A and C production. This suggests that endogenous factors influence surfactin production. Therefore, we aimed to identify the genetic factor that regulates surfactin congener production. The srfA operon for surfactin A biosynthesis in B. subtilis , composed of four open reading frames (ORFs), is srfAABCD . Comparative genomic analysis between the B. subtilis JCM 1465 srfA operon and the TUA12 surfactin biosynthesis genes examined in this study revealed that the operon responsible for surfactin A biosynthesis is distinct, exhibiting 68.7%, 69.2%, 84.7%, and 67.4% homology with the four ORFs, respectively. Similarly, the operon for Ptrs2 surfactin C biosynthesis showed 68.7%, 69.2%, 64.4%, and 67.1% homology. These differences indicate that the identified surfactin A and C biosynthetic operons are novel genetic variants. Further analysis identified the adenylation domain responsible for selecting Ile7 in surfactin C via domain substitution in a surfactin A-producing strain. Average nucleotide identity analysis showed that the surfactin A and C operons were found in B. velezensis and B. amyloliquefaciens , respectively. Our findings suggest that surfactin congener production is species-dependent, with the srf operon specifically distributed in Bacillus spp.

An artifact of recombinatorial cloning challenges established beliefs of plasmid cotransformation, selection, and maintenance

2025-05-27

Courtney L Geer , Michael Charette

Gateway cloning is an easy, efficient, accurate, and versatile cloning strategy. During Expression clone validation, we sometimes see an additional band co-migrating with the pDONR (Entry) backbone. We show that … Gateway cloning is an easy, efficient, accurate, and versatile cloning strategy. During Expression clone validation, we sometimes see an additional band co-migrating with the pDONR (Entry) backbone. We show that this “mystery” band is not an artifact of aberrant recombination but instead originates from a cotransformation event, where more than one different plasmid is transformed into a single E. coli cell simultaneously and in the absence of antibiotic selection. We find that the unselected pDONR Entry plasmid is cotransformed into E. coli with the desired Expression vector in 9–29% of colonies and is maintained without antibiotic selection, despite plasmid incompatibility. We propose an easy strategy to screen for and eliminate cotransformants. Our results challenge accepted beliefs of bacterial plasmid transformation, selection, and maintenance and comprise the first documented occurrence of cotransformation in Gateway cloning.

Time-resolved analysis of Bacillus subtilis DB104 Spo0A-mutant transcriptome profile and enhancement of recombinant protein release

2025-05-27

Ji-Su Jun , Soo Ji Kang , Kwang‐Won Hong

Cryo-EM Structure of the relaxosome, a complex essential for bacterial mating and the spread of antibiotic resistance genes

2025-05-27

Sunanda Margrett Williams , Sandra Raffl , Sabine Kienesberger +3 more

Substrate-induced assembly and functional mechanism of the bacterial membrane protein insertase SecYEG-YidC

2025-05-27

M. Busch , Cristian Rosales , Michael Kamel +6 more

The universally conserved Sec translocon and the YidC/Oxa1-type insertases mediate biogenesis of alpha-helical membrane proteins, but the molecular basis of their cooperation has remained disputed over decades. A recent discovery … The universally conserved Sec translocon and the YidC/Oxa1-type insertases mediate biogenesis of alpha-helical membrane proteins, but the molecular basis of their cooperation has remained disputed over decades. A recent discovery of a multi-subunit insertase in eukaryotes has raised the question about the architecture of the putative bacterial ortholog SecYEG-YidC and its functional mechanism. Here, we combine cryogenic electron microscopy with cell-free protein synthesis in nanodiscs to visualize biogenesis of the polytopic membrane protein NuoK, the subunit K of NADH-quinone oxidoreductase, that requires both SecYEG and YidC for insertion. We demonstrate that YidC is recruited to the back of the translocon at the late stage of the substrate insertion, in resemblance to the eukaryotic system, and in vivo experiments indicate that the complex assembly is vital for the cells. The nascent chain does not utilize the lateral gate of SecYEG, but enters the lipid membrane at the SecYE-YidC interface, with YidC being the primary insertase. SecYEG-YidC complex promotes folding of the nascent helices at the interface prior their insertion, so the examined cellular pathway follows the fundamental thermodynamic principles of membrane protein folding. Our data provide the first detailed insight on the elusive insertase machinery in the physiologically relevant environment, highlight the importance of the nascent chain for its assembly, and prove the evolutionary conservation of the gate-independent insertion route.

The bacterial transcription terminator, Rho, functions as an RNA:DNA hybrid (RDH) helicase in vivo.

2025-05-26

A.V. Bhosale , Ranjan Sen

Ribonuclease HI (rnhA) removes the deleterious RNA:DNA hybrids (RDHs) by cleaving its RNA component. The bacterial transcription terminator Rho is an RNA-dependent 5' → 3' helicase capable of unwinding RDH … Ribonuclease HI (rnhA) removes the deleterious RNA:DNA hybrids (RDHs) by cleaving its RNA component. The bacterial transcription terminator Rho is an RNA-dependent 5' → 3' helicase capable of unwinding RDH formed on a single-stranded RNA in vitro. We hypothesize that Rho might be directly involved in RDH removal in vivo. Here, we demonstrate that Rho primary RNA-binding site (PBS) mutants defective in RNA binding and helicase activity are synthetically lethal specifically when RNase HI is absent. This lethality was not observed in the absence of RNase HII (rnhB) alone. Rho-PBS mutants in an rnhA- strain exhibited increased plasmid-concatemer and plasmid copy number, altered cell morphology, and were highly susceptible to DNA-damaging agents. These Rho mutants increased the accumulation of RDHs in vivo, suggesting defects in the RDH removal process. Rho was colocalized to RDHs in vivo when RNase HI was absent. Certain catalytically inactive mutants of RNase H that bind to the RDH blocked the entry of Rho to the RDH, inducing cell death, indicating the role of Rho in the removal of deleterious RDHs in the absence of RNase HI. Under in vitro conditions, Rho was capable of binding to the RDHs and unwinding them in a rut-site-dependent manner. Therefore, we concluded that in the absence of RNase HI, Rho, by its RNA-dependent helicase activity, is capable of unwinding RDHs in a rut-site-dependent manner. These results establish the non-transcription terminator role of Rho and its functional synergy with RNase HI in vivo.