Systematic Inactivation and Phenotypic Characterization of Two-Component Signal Transduction Systems of Enterococcus faecalis V583

Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Journal of Bacteriology (Impact Factor: 2.81). 01/2005; 186(23):7951-8. DOI: 10.1128/JB.186.23.7951-7958.2004
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The ability of enterococci to adapt and respond to different environmental stimuli, including the host environment, led us
to investigate the role of two-component signal transduction in the regulation of Enterococcus faecalis physiology. Using a bioinformatic approach, we previously identified 17 two-component systems (TCS), consisting of a sensory
histidine kinase and the cognate response regulator, as well as an additional orphan response regulator (L. E. Hancock and
M. Perego, J. Bacteriol. 184:5819-5825, 2002). In an effort to identify the potential function of each TCS in the biology
of E. faecalis clinical isolate strain V583, we constructed insertion mutations in each of the response regulators. We were able to inactivate
17 of 18 response regulators, the exception being an ortholog of YycF, previously shown to be essential for viability in a
variety of gram-positive microorganisms. The biological effects of the remaining mutations were assessed by using a number
of assays, including antibiotic resistance, biofilm formation, and environmental stress. We identified TCS related to antibiotic
resistance and environmental stress and found one system which controls the initiation of biofilm development by E. faecalis.

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Available from: Lynn Hancock, Jun 15, 2015
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    • "Among all the transcriptional regulators found in the E. mundtii genome, there is a subset corresponding to response regulators (RR) putatively forming part of two-component signal transduction systems (TCS). The importance of TCS for the ability of E. faecalis to respond to environmental stimuli has been previously stressed by Hancock and Perego [55]. The E. mundtii genome contains putative genes coding for 17 TCS and one orphan response regulator (Additional file 4: Table S3). "
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    ABSTRACT: Background: Enterococcus mundtii is a yellow-pigmented microorganism rarely found in human infections. The draft genome sequence of E. mundtii was recently announced. Its genome encodes at least 2,589 genes and 57 RNAs, and 4 putative genomic islands have been detected. The objective of this study was to compare the genetic content of E. mundtii with respect to other enterococcal species and, more specifically, to identify genes coding for putative virulence traits present in enterococcal opportunistic pathogens. Results: An in-depth mining of the annotated genome was performed in order to uncover the unique properties of this microorganism, which allowed us to detect a gene encoding the antimicrobial peptide mundticin among other relevant features. Moreover, in this study a comparative genomic analysis against commensal and pathogenic enterococcal species, for which genomic sequences have been released, was conducted for the first time. Furthermore, our study reveals significant similarities in gene content between this environmental isolate and the selected enterococci strains (sharing an "enterococcal gene core" of 805 CDS), which contributes to understand the persistence of this genus in different niches and also improves our knowledge about the genetics of this diverse group of microorganisms that includes environmental, commensal and opportunistic pathogens. Conclusion: Although E. mundtii CRL1656 is phylogenetically closer to E. faecium, frequently responsible of nosocomial infections, this strain does not encode the most relevant relevant virulence factors found in the enterococcal clinical isolates and bioinformatic predictions indicate that it possesses the lowest number of putative pathogenic genes among the most representative enterococcal species. Accordingly, infection assays using the Galleria mellonella model confirmed its low virulence.
    BMC Genomics 06/2014; 15(1):489. DOI:10.1186/1471-2164-15-489 · 3.99 Impact Factor
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    • "Although the WalRK TCS shows distinctive features in each bacterial species, several unifying patterns have emerged about this TCS. The WalR response regulator is generally essential and required for growth in culture (Fabret and Hoch, 1998; Martin et al., 1999; Throup et al., 2000; Wagner et al., 2002; Kadioglu et al., 2003; Hancock and Perego, 2004; Senadheera et al., 2005), although bypass suppressors can be identified that uncouple regulon expression from WalR control (Ng et al., 2003; Winkler and Hoch, 2008; Delaune et al., 2011). WalR proteins are members of the PhoB family of response regulators and consist of phosphorylated receiver domains and winged-helix DNA-binding effector domains that contain characteristic structural features (see Bent et al., 2004; Okajima et al., 2008; Barbieri et al., 2010; Doi et al., 2010). "
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    ABSTRACT: WalRK (YycFG) two-component systems (TCSs) of low-GC Gram-positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low-GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR∼P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR∼P amounts in cells determined by Phos-tag SDS-PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR∼P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross-talk and the recovery of WalR∼P from walK(+) cells.
    Molecular Microbiology 09/2012; 86(3):645-60. DOI:10.1111/mmi.12006 · 4.42 Impact Factor
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    • "Plasmid pJFP76 was constructed by amplifying the tetM cassette of pJM133 (kindly provided by M. Perego, Scripps Research Institute) (Hancock & Perego, 2004) with primers Tet-NotI-up/Tet-NcoI-dn, and ligating the digested fragment into the NcoI and NotI restriction sites of pJFP46. Transformation of SK36 with pJFP76 produced the Tet-resistant strain JFP76. "
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    ABSTRACT: Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo.
    Microbiology 06/2009; 155(Pt 8):2573-82. DOI:10.1099/mic.0.024513-0 · 2.56 Impact Factor
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