scholarly journals Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea

2012 ◽  
Vol 80 (4) ◽  
pp. 1323-1332 ◽  
Author(s):  
Ayaka Shima ◽  
Atsushi Hinenoya ◽  
Masahiro Asakura ◽  
Norihiko Sugimoto ◽  
Teizo Tsukamoto ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Eukaryotic Cell ◽  
Nucleotide Sequence Analysis ◽  
M Cell ◽  
Transposase Gene ◽  
Content Type ◽  
Histone H2ax ◽  
E Coli ◽  
Shigella Boydii ◽  
Providencia Alcalifaciens

ABSTRACTCytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides—CdtA, CdtB, and CdtC—encoded by the genescdtA,cdtB, andcdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles ofcdtB(Cdt-I through Cdt-V) inEscherichia coliand used the assay to investigate the prevalence and characteristic of CDT-producingE. coliin children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206–215, 2009). In these assays, two untypablecdtBgenes were detected and the organisms harboring thecdtBgene were identified asProvidencia alcalifaciens(strains AH-31 and AS-1). Nucleotide sequence analysis of thecdtgene cluster revealed that thecdtA,cdtB, andcdtCgenes ofP. alcalifaciensare of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of thecdtgenes among closely related species, the presence ofcdtgenes was screened in variousProvidenciaspp. by colony hybridization assay, and thecdtgene cluster was found in only limited strains ofP. alcalifaciens. Genome walking revealed that thecdtgene cluster ofP. alcalifaciensis located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT ofP. alcalifaciens(PaCDT) showed some homology with the CDT ofShigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells,E. coliCDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G2/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinicalP. alcalifaciensstrains could produce variants of the CDTs compared.

scholarly journals A Bacterial Microcompartment Is Used for Choline Fermentation byEscherichia coli536

2018 ◽  
Vol 200 (10) ◽  
Author(s):  
Taylor I. Herring ◽  
Tiffany N. Harris ◽  
Chiranjit Chowdhury ◽  
Sujit Kumar Mohanty ◽  
Thomas A. Bobik
Keyword(s):  
Electron Microscopy ◽  
Heart Disease ◽  
Gene Cluster ◽  
Transcriptional Regulator ◽  
Human Gut ◽  
Content Type ◽  
E Coli ◽  
Bacterial Microcompartment ◽  
New Type ◽  
Insight Into

ABSTRACTBacterial choline degradation in the human gut has been associated with cancer and heart disease. In addition, recent studies found that a bacterial microcompartment is involved in choline utilization byProteusandDesulfovibriospecies. However, many aspects of this process have not been fully defined. Here, we investigate choline degradation by the uropathogenEscherichia coli536. Growth studies indicatedE. coli536 degrades choline primarily by fermentation. Electron microscopy indicated that a bacterial microcompartment was used for this process. Bioinformatic analyses suggested that the choline utilization (cut) gene cluster ofE. coli536 includes two operons, one containing three genes and a main operon of 13 genes. Regulatory studies indicate that thecutXgene encodes a positive transcriptional regulator required for induction of the maincutoperon in response to choline supplementation. Each of the 16 genes in thecutcluster was individually deleted, and phenotypes were examined. ThecutX,cutY,cutF,cutO,cutC,cutD,cutU, andcutVgenes were required for choline degradation, but the remaining genes of thecutcluster were not essential under the conditions used. The reasons for these varied phenotypes are discussed.IMPORTANCEHere, we investigate choline degradation inE. coli536. These studies provide a basis for understanding a new type of bacterial microcompartment and may provide deeper insight into the link between choline degradation in the human gut and cancer and heart disease. These are also the first studies of choline degradation inE. coli536, an organism for which sophisticated genetic analysis methods are available. In addition, thecutgene cluster ofE. coli536 is located in pathogenicity island II (PAI-II536) and hence might contribute to pathogenesis.

scholarly journals Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

2020 ◽  
Vol 202 (10) ◽  
Author(s):  
Yannick R. Brunet ◽  
Christophe S. Bernard ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Secretion System ◽  
Target Cells ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Internal Promoter ◽  
E Coli ◽  
Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

scholarly journals Comparative Analysis of EspF Variants in Inhibition of Escherichia coli Phagocytosis by Macrophages and Inhibition of E. coli Translocation through Human- and Bovine-Derived M Cells

2011 ◽  
Vol 79 (11) ◽  
pp. 4716-4729 ◽  
Author(s):  
Amin Tahoun ◽  
Gabriella Siszler ◽  
Kevin Spears ◽  
Sean McAteer ◽  
Jai Tree ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cellular Localization ◽  
M Cells ◽  
Binding Assays ◽  
M Cell ◽  
Content Type ◽  
E Coli ◽  
Coculture System

ABSTRACTThe EspF protein is secreted by the type III secretion system of enteropathogenic and enterohemorrhagicEscherichia coli(EPEC and EHEC, respectively). EspF sequences differ between EHEC O157:H7, EHEC O26:H11, and EPEC O127:H6 in terms of the number of SH3-binding polyproline-rich repeats and specific residues in these regions, as well as residues in the amino domain involved in cellular localization. EspFO127is important for the inhibition of phagocytosis by EPEC and also limits EPEC translocation through antigen-sampling cells (M cells). EspFO127has been shown to have effects on cellular organelle function and interacts with several host proteins, including N-WASP and sorting nexin 9 (SNX9). In this study, we compared the capacities of differentespFalleles to inhibit (i) bacterial phagocytosis by macrophages, (ii) translocation through an M-cell coculture system, and (iii) uptake by and translocation through cultured bovine epithelial cells. TheespFgene fromE. coliserotype O157 (espFO157) allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibitE. colitranslocation through a human-derivedin vitroM-cell coculture system in comparison toespFO127andespFO26. In contrast,espFO157was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonization site of EHEC O157 in cattle and a site containing M-like cells. Although LUMIER binding assays demonstrated differences in the interactions of the EspF variants with SNX9 and N-WASP, we propose that other, as-yet-uncharacterized interactions contribute to the host-based variation in EspF activity demonstrated here.

scholarly journals AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

Microbiology ◽  
2020 ◽  
Vol 166 (6) ◽  
pp. 567-578 ◽  
Author(s):  
Fariza Ammam ◽  
Delphine Patin ◽  
Héloise Coullon ◽  
Didier Blanot ◽  
Thierry Lambert ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Gene Cluster ◽  
Enterococcus Faecalis ◽  
Type Species ◽  
Asparagine Synthetase ◽  
Structure Modification ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Peptidoglycan Structure

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

scholarly journals Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

2016 ◽  
Vol 54 (4) ◽  
pp. 1074-1081 ◽  
Author(s):  
Masahiro Kusumoto ◽  
Yuna Hikoda ◽  
Yuki Fujii ◽  
Misato Murata ◽  
Hirotsugu Miyoshi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Significant Risk ◽  
Multidrug Resistant ◽  
Limited Range ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Shigella Boydii ◽  
High Level

EnterotoxigenicEscherichia coli(ETEC) and Shiga toxin-producingE. coli(STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenicE. colistrains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenicE. coli. In the present study, we determined the O serogroups of 967E. coliisolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup ofShigella boydiitype 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

scholarly journals Exopolysaccharide Production and Ropy Phenotype Are Determined by Two Gene Clusters in Putative Probiotic Strain Lactobacillus paraplantarum BGCG11

2014 ◽  
Vol 81 (4) ◽  
pp. 1387-1396 ◽  
Author(s):  
Milica Zivkovic ◽  
Marija Miljkovic ◽  
Patricia Ruas-Madiedo ◽  
Ivana Strahinic ◽  
Maja Tolinacki ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gene Cluster ◽  
Probiotic Strain ◽  
Gene Clusters ◽  
Size Exclusion ◽  
High Molecular Weight Fraction ◽  
Transposase Gene ◽  
Content Type ◽  
Lactobacillus Paraplantarum

ABSTRACTLactobacillus paraplantarumBGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high-molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of theepsgene cluster, a putative transposase gene was identified, followed by an additionalrfbgene cluster containing therfbACBDgenes, the ones most probably responsible for dTDP-l-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in theepsand the other in therfbgene cluster, as confirmed by size exclusion chromatography analysis. Therefore, bothepsandrfbgenes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strainL. paraplantarumBGCG11.

scholarly journals TheEscherichia coliType III Secretion System 2 Has a Global Effect on Cell Surface

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alexander Shulman ◽  
Yael Yair ◽  
Dvora Biran ◽  
Thomas Sura ◽  
Andreas Otto ◽  
...  
Keyword(s):  
Cell Surface ◽  
Gene Cluster ◽  
Type Iii Secretion ◽  
Membrane Vesicles ◽  
Secretion System ◽  
Outer Membrane Vesicles ◽  
Content Type ◽  
E Coli ◽  
Flagellar Proteins ◽  
Tract Infections

ABSTRACTMany strains ofEscherichia colicarry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to theSalmonellapathogenicity island 1 (SPI-1) system, encoding a T3SS inSalmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked inE. coliserotype O2—associated with urinary tract infections and septicemia—which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis.IMPORTANCEDrug-resistant extraintestinal pathogenicE. coli(ExPEC) strains are major pathogens, especially in hospital- and community-acquired infections. They are the major cause of urinary tract infections and are often involved in septicemia with high mortality. ExPEC strains are characterized by broad-spectrum antibiotic resistance, and development of a vaccine is not trivial because the ExPEC strains include a large number of serotypes. It is therefore important to understand the virulence factors that are involved in pathogenicity of ExPEC and identify new targets for development of antibacterial drugs or vaccines. Such a target could be ETT2, a unique type III secretion system present (complete or in parts) in many ExPEC strains. Here, we show that this system has a major effect on the bacterial surface—it affects sensitivity to drugs, motility, and secretion of extracellular proteins and outer membrane vesicles. Most importantly, this system is important for serum resistance, a prerequisite for septicemia.

scholarly journals Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli

2016 ◽  
Vol 82 (20) ◽  
pp. 6167-6173 ◽  
Author(s):  
Meenu Katoch ◽  
Rabia Mazmouz ◽  
Rocky Chau ◽  
Leanne A. Pearson ◽  
Russell Pickford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Uv Radiation ◽  
Active Ingredient ◽  
Stress Factors ◽  
Heterologous Production ◽  
Content Type ◽  
E Coli

ABSTRACTMycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacteriumCylindrospermum stagnalePCC 7417 revealed a new gene cluster with homology to MAA synthase fromNostoc punctiforme. This newly identified gene cluster is unusual because it has five biosynthesis genes (mylAtomylE), compared to the four found in other MAA gene clusters. Heterologous expression ofmylAtomylEinEscherichia coliresulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced inE. coliand structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods.IMPORTANCEMycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs inE. coliis also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

scholarly journals Evaluation of a Culture-Dependent Algorithm and a Molecular Algorithm for Identification ofShigellaspp.,Escherichia coli, and EnteroinvasiveE. coli

2018 ◽  
Vol 56 (10) ◽  
Author(s):  
Maaike J. C. van den Beld ◽  
Richard F. de Boer ◽  
Frans A. G. Reubsaet ◽  
John W. A. Rossen ◽  
Kai Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shigella Flexneri ◽  
Clinical Diagnostics ◽  
Shigella Dysenteriae ◽  
Identification Algorithm ◽  
Content Type ◽  
E Coli ◽  
Shigella Boydii ◽  
Discrepancy Analysis ◽  
Culture Dependent

ABSTRACTIdentification ofShigellaspp.,Escherichia coli, and enteroinvasiveE. coli(EIEC) is challenging because of their close relatedness. Distinction is vital, as infections withShigellaspp. are under surveillance of health authorities, in contrast to EIEC infections. In this study, a culture-dependent identification algorithm and a molecular identification algorithm were evaluated. Discrepancies between the two algorithms and original identification were assessed using whole-genome sequencing (WGS). After discrepancy analysis with the molecular algorithm, 100% of the evaluated isolates were identified in concordance with the original identification. However, the resolution for certain serotypes was lower than that of previously described methods and lower than that of the culture-dependent algorithm. Although the resolution of the culture-dependent algorithm is high, 100% of noninvasiveE. coli,Shigella sonnei, andShigella dysenteriae, 93% ofShigella boydiiand EIEC, and 85% ofShigella flexneriisolates were identified in concordance with the original identification. Discrepancy analysis using WGS was able to confirm one of the used algorithms in four discrepant results. However, it failed to clarify three other discrepant results, as it added yet another identification. Both proposed algorithms performed well for the identification ofShigellaspp. and EIEC isolates and are applicable in low-resource settings, in contrast to previously described methods that require WGS for daily diagnostics. Evaluation of the algorithms showed that both algorithms are capable of identifyingShigellaspecies and EIEC isolates. The molecular algorithm is more applicable in clinical diagnostics for fast and accurate screening, while the culture-dependent algorithm is more suitable for reference laboratories to identifyShigellaspp. and EIEC up to the serotype level.

scholarly journals A Modular, Tn7-Based System for Making Bioluminescent or Fluorescent Salmonella and Escherichia coli Strains

2016 ◽  
Vol 82 (16) ◽  
pp. 4931-4943 ◽  
Author(s):  
Dylan J. Shivak ◽  
Keith D. MacKenzie ◽  
Nikole L. Watson ◽  
J. Alex Pasternak ◽  
Brian D. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
High Throughput ◽  
Constitutive Expression ◽  
Bacterial Strains ◽  
Efficient System ◽  
Transposase Gene ◽  
Content Type ◽  
E Coli

ABSTRACTOur goal was to develop a robust tagging method that can be used to track bacterial strainsin vivo. To address this challenge, we adapted two existing systems: a modular plasmid-based reporter system (pCS26) that has been used for high-throughput gene expression studies inSalmonellaandEscherichia coliand Tn7transposition. We generated kanamycin- and chloramphenicol-resistant versions of pCS26 with bacterial luciferase, green fluorescent protein (GFP), and mCherry reporters under the control of σ70-dependent promoters to provide three different levels of constitutive expression. We improved upon the existing Tn7system by modifying the delivery vector to accept pCS26 constructs and moving the transposase genes from a nonreplicating helper plasmid into a temperature-sensitive plasmid that can be conditionally maintained. This resulted in a 10- to 30-fold boost in transposase gene expression and transposition efficiencies of 10−8to 10−10inSalmonella entericaserovar Typhimurium andE. coliAPEC O1, whereas the existing Tn7system yielded no successful transposition events. The new reporter strains displayed reproducible signaling in microwell plate assays, confocal microscopy, andin vivoanimal infections. We have combined two flexible and complementary tools that can be used for a multitude of molecular biology applications within theEnterobacteriaceae. This system can accommodate new promoter-reporter combinations as they become available and can help to bridge the gap between modern, high-throughput technologies and classical molecular genetics.IMPORTANCEThis article describes a flexible and efficient system for tagging bacterial strains. Using our modular plasmid system, a researcher can easily change the reporter type or the promoter driving expression and test the parameters of these new constructsin vitro. Selected constructs can then be stably integrated into the chromosomes of desired strains in two simple steps. We demonstrate the use of this system inSalmonellaandE. coli, and we predict that it will be widely applicable to other bacterial strains within theEnterobacteriaceae. This technology will allow for improvedin vivoanalysis of bacterial pathogens.

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