Functional Genomics of<i>Enterococcus faecalis</i>: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

Ballering, Katie S.; Kristich, Christopher J.; Grindle, Suzanne; Oromendia, Ana B.; Beattie, David T.; Dunny, Gary M.

doi:10.1128/jb.01688-08

Cited by 57 publications

(73 citation statements)

References 60 publications

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“…To test the hypothesis that enterococcal biofilm determinants are virulence factors in endocarditis, we chose seven loci (Table 1) in the chromosome of E. faecalis OG1RF that were associated with biofilm formation in our previous studies (20,25). The following five loci were disrupted with transposon insertions: EF0676, EF0799, EF0983, EF0999, and EF1718.…”

Section: Resultsmentioning

confidence: 99%

“…The in-frame markerless deletion of the recN (EF0984) locus was previously described in an OG1RF(pCF10) strain (25). The same constructs and procedures were used in this study to generate a pCF10-free version of OG1RF ⌬recN.…”

Section: Methodsmentioning

confidence: 99%

“…No common genes were found between the transposon and biofilm RIVET screens, but the screens were complementary in that there were four genomic regions with two adjacent genes where one gene was identified in the RIVET screen and the other gene was identified in the transposon screen. Two genes with transposon insertions encode transcriptional regulators, suggesting the possibility of regulatory interactions between these and the adjacent genes that were identified in the RIVET screen (25). The ability to form biofilms is common among E. faecalis isolates of clinical origin (26,27), but a broad analysis to identify the genetic loci on the E. faecalis chromosome that are responsible for biofilm-associated infection has not been undertaken.…”

mentioning

confidence: 99%

“…To that end, we tested whether five of the biofilm-defective transposon insertion mutants (20) were attenuated in a rabbit model of endocarditis. In addition, we sought to test whether the complementary outcome of the combined results of our previous transposon and RIVET screens predicted regulatory interactions among biofilm-associated genes (25). To do this, we characterized the biofilm formation and endocarditis virulence phenotypes of two strains with in-frame deletions of genes identified in our RIVET screen that are adjacent to the two biofilm-associated ArgR family transcriptional regulators identified in the transposon screen.…”

mentioning

confidence: 99%

“…To the best of our knowledge, the other seven loci have not been studied directly in virulence models. We also used recombinase-based in vivo expression technology (RIVET) to identify promoters that are upregulated in E. faecalis OG1RF during in vitro biofilm growth (25). No common genes were found between the transposon and biofilm RIVET screens, but the screens were complementary in that there were four genomic regions with two adjacent genes where one gene was identified in the RIVET screen and the other gene was identified in the transposon screen.…”

mentioning

confidence: 99%

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AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

et al. 2013

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bEnterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

et al. 2013

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Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry

et al. 2016

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Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC ‐ ESI mass spectrometry. We identified 222 membrane‐associated proteins, which represent approximately 24% of the predicted membrane‐associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane‐associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment.

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Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR

Saavedra

Schwartzman

Gilmore

2017

CP Molecular Biology

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Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to i) amplify DNA spanning the transposon insertion junction, and ii) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2–3 days if starting from a culture of the transposon insertion mutant.

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Functional Genomics ofEnterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

Cited by 57 publications

References 60 publications

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

AhrC and Eep Are Biofilm Infection-Associated Virulence Factors in Enterococcus faecalis

Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry

Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR

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