Two Nonredundant SecA Homologues Function in Mycobacteria

ABSTRACT The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis ofMycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export—the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy inMycobacterium smegmatis, we demonstrate thatsecA1 is an essential gene. In contrast,secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature ofsecA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a ΔsecA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.

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Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

Journal of Bacteriology ◽

10.1128/jb.185.9.2811-2819.2003 ◽

2003 ◽

Vol 185 (9) ◽

pp. 2811-2819 ◽

Cited By ~ 62

Author(s):

Natascha Blaudeck ◽

Peter Kreutzenbeck ◽

Roland Freudl ◽

Georg A. Sprenger

Keyword(s):

Escherichia Coli ◽

Signal Peptide ◽

Protein Translocation ◽

Alternative Possibilities ◽

Amino Acid Residues ◽

Sec Pathway ◽

Twin Arginine Translocation ◽

Tat Pathway ◽

Arginine Residues ◽

Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

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The E. coli ffh gene is necessary for viability and efficient protein export

Nature ◽

10.1038/359744a0 ◽

1992 ◽

Vol 359 (6397) ◽

pp. 744-746 ◽

Cited By ~ 172

Author(s):

Gregory J. Phillips ◽

Thomas J. Silhavy

Keyword(s):

Protein Export ◽

E Coli

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Mechanistic Challenges and Engineering Applications of Protein Export in E. coli

Systems Biology and Biotechnology of Escherichia coli ◽

10.1007/978-1-4020-9394-4_16 ◽

2009 ◽

pp. 327-349

Author(s):

Eva-Maria Strauch ◽

George Georgiou

Keyword(s):

Protein Export ◽

Engineering Applications ◽

E Coli

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Genomic and phenotypic analysis of heat and sanitizer resistance in Escherichia coli from beef in relation to the locus of heat resistance

Applied and Environmental Microbiology ◽

10.1128/aem.01574-21 ◽

2021 ◽

Author(s):

Xianqin Yang ◽

Frances Tran ◽

Peipei Zhang ◽

Hui Wang

Keyword(s):

Escherichia Coli ◽

Heat Resistance ◽

Phylogenetic Relationships ◽

Core Genome ◽

Elevated Temperatures ◽

Resistant Bacteria ◽

Phenotypic Analysis ◽

Decimal Reduction Time ◽

E Coli ◽

Clade 1

The locus of heat resistance (LHR) can confer heat resistance to Escherichia coli to various extents. This study investigated the phylogenetic relationships, and genomic and phenotypic characteristics of E. coli with or without LHR recovered from beef by direct plating or from enrichment broth at 42°C. LHR-positive E. coli isolates (n=24) were whole genome-sequenced by short- and long-reads. LHR-negative isolates (n=18) from equivalent sources as LHR-positive isolates were short-read sequenced. All isolates were assessed for decimal reduction time at 60°C ( D 60°C ) and susceptibility to E-SAN and Perox-E. Selected isolates were evaluated for growth at 42°C. The LHR-positive and negative isolates were well separated on the core genome tree, with 22/24 of the positive isolates clustering into three clades. Isolates within clade 1 and 2, despite their different D 60°C values, were clonal, as determined by subtyping (MLST, core genome MLST, and serotyping). Isolates within each clade are of one serotype. The LHR-negative isolates were genetically diverse. The LHR-positive isolates had a larger (p<0.001) median genome size by 0.3 Mbp (5.0 vs 4.7 Mbp), and overrepresentation of genes in plasmid maintenance, stress response and cryptic prophages, but underrepresentation of genes involved in epithelial attachment and virulence. All LHR-positive isolates harbored a chromosomal copy of LHR, and all clade 2 isolates had an additional partial copy of LHR on conjugative plasmids. The growth rates at 42°C were 0.71±0.02 and 0.65±0.02 logOD h −1 for LHR-positive and negative isolates. No meaningful difference in sanitizer susceptibility was noted between LHR-positive and negative isolates. Importance Resistant bacteria are serious food safety and public health concerns. Heat resistance conferred by the LHR varies largely among different strains. The findings in this study show that genomic background and composition of LHR, in addition to the presence of LHR, play an important role in the degree of heat resistance in E. coli , and that strains with certain genetic background are more likely to acquire and maintain the LHR. Also, caution should be exercised when recovering E. coli at elevated temperatures as the presence of LHR may confer growth advantages to some strains. Interestingly, the LHR harboring strains seem to have evolved further from their primary animal host to adapt to their secondary habitat, as reflected by fewer genes in virulence and epithelial attachment. The phylogenetic relationships among the isolates point towards multiple mechanisms for acquiring LHR, likely prior to their deposition on meat.

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Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35–38 Amino Acids of GyrA

Microorganisms ◽

10.3390/microorganisms7030081 ◽

2019 ◽

Vol 7 (3) ◽

pp. 81 ◽

Cited By ~ 2

Author(s):

Nikolay Rovinskiy ◽

Andrews Agbleke ◽

Olga Chesnokova ◽

N. Higgins

Keyword(s):

Essential Gene ◽

Wild Type ◽

Chromosomal Dna ◽

E Coli ◽

Sister Chromosome ◽

Negative Supercoiling ◽

Close Relatives ◽

Negative Supercoils

Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.

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The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The EMBO Journal ◽

10.1002/j.1460-2075.1994.tb06703.x ◽

1994 ◽

Vol 13 (16) ◽

pp. 3917-3924 ◽

Cited By ~ 38

Author(s):

C. Rasmussen ◽

C. Garen ◽

S. Brining ◽

R.L. Kincaid ◽

R.L. Means ◽

...

Keyword(s):

Aspergillus Nidulans ◽

Protein Phosphatase ◽

Essential Gene ◽

Catalytic Subunit ◽

Calcineurin A ◽

Dependent Protein

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Antimicrobial Resistance Profiles and Characterization of Escherichia coli Strains from Cases of Neonatal Diarrhea in Spanish Pig Farms

Veterinary Sciences ◽

10.3390/vetsci7020048 ◽

2020 ◽

Vol 7 (2) ◽

pp. 48

Author(s):

Anna Vidal ◽

Laia Aguirre ◽

Chiara Seminati ◽

Montse Tello ◽

Noelia Redondo ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Animal Health ◽

Phenotypic Analysis ◽

Low Frequencies ◽

Neonatal Diarrhea ◽

E Coli ◽

Pig Farms ◽

Diagnostic Approaches ◽

Low Prevalence

Escherichia coli is considered one of the most common agents associated with neonatal diarrhea in piglets. The aim of this work was to characterize the pathogenic and antimicrobial resistance (AMR) profiles of 122 E. coli strains isolated from pigs suffering diarrhea (n = 94) and pigs without diarrhea (n = 28) of 24 farms in Spain. Virulence factors, toxins and AMR (ESBL and colistin) genes and AMR phenotypes of E. coli isolates were analyzed. Low prevalence of pathogenic E. coli strains (26%) was found in both groups. However, ETEC and VTEC strains were more frequently isolated from diarrheic piglets. Irrespectively of diarrhea occurrence, 97.5% of the strains showed a multidrug-resistance (MDR) profile to aminopenicillins, sulfonamides and tetracyclines. It was found that 22% of E. coli was CTX-M+, with CTX-M-14 being the principal allelic variant. Remarkably, 81.5% of CTX-M+ strains were isolated from diarrheic animals and presented an extended MDR profile to aminopenicillins, quinolones and aminoglycosides. Finally, low frequencies of colistin resistance genes mcr-1 (4/122) and mcr-4 (1/122) were found. MDR E. coli strains are circulating in pig farms of Spain, representing a serious threat to animal and public health. More appropriate diagnostic approaches (genetic and AMR phenotypic analysis) should be implemented in animal health to optimize antibiotic treatments.

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Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Viruses ◽

10.3390/v12111247 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1247

Author(s):

Athina Zampara ◽

Stephen J. Ahern ◽

Yves Briers ◽

Lone Brøndsted ◽

Martine Camilla Holst Sørensen

Keyword(s):

Signal Peptide ◽

In Silico ◽

Protein Domains ◽

In Silico Analysis ◽

Cell Lysis ◽

E Coli ◽

Sec Pathway ◽

Gene Functions ◽

Lytic Genes ◽

Silico Analysis

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

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Slow Polymerization of Mycobacterium tuberculosis FtsZ

Journal of Bacteriology ◽

10.1128/jb.182.14.4028-4034.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 4028-4034 ◽

Cited By ~ 92

Author(s):

E. Lucile White ◽

Larry J. Ross ◽

Robert C. Reynolds ◽

Lainne E. Seitz ◽

Georgia D. Moore ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Tubulin Polymerization ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Minimum Concentration ◽

E Coli ◽

Cell Division Protein ◽

Mycobacterial Protein

ABSTRACT The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coliand purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than E. coliFtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg2+and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

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