Systematic inactivation and phenotypic characterization of two-component signal transduction systems of Enterococcus faecalis V583

Abstract

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.

FIG. 1.

Schematic of insertional mutagenesis of E. faecalis V583 response regulators. (A) Construction of p3TET insertion vector. Vector p3ERM was restricted with MfeI and NaeI, and the larger 1.85-kb fragment was ligated to the tetM gene (EcoRI/HincII) from pJM133. (B) Internal gene fragments from each response regulator were cloned into p3TET, and the resulting constructs were used to transform E. faecalis V583. Insertions were confirmed by colony PCR using primers flanking the insertion site along with primers corresponding to the insertion vector.

FIG. 2.

Growth curves of E. faecalis strains. Cells were grown in THB at 37°C with growth monitored by OD₆₀₀. Filled diamonds, V583T; filled triangles, RR04; filled squares, RR14. Error bars denote standard deviations.

FIG. 3.

Growth curves of E. faecalis strains in the presence of erythromycin. Cells were grown in THB with erythromycin (256 μg/ml) at 37°C with growth monitored by OD₆₀₀. Filled circles, V583T; open squares, RR02; filled triangles, RR11; filled squares, RR14; filled diamonds, RR15; open circles, RR16; open triangles, RR18. Error bars denote standard deviations.

FIG. 4.

Growth curves of E. faecalis strains with environmental stress. (A) Growth of E. faecalis strains at 46°C were monitored at OD₅₅₀ using a microtiter plate reader. Filled circles, V583T; filled squares, RR06. (B) Growth curves of E. faecalis strains in THB with SDS (0.003%) at 37°C with growth monitored at OD₆₀₀. Filled circles, V583T; filled squares, RR06. Error bars denote standard deviations.

FIG. 5.

Biofilm formation by E. faecalis V583T and response regulator mutants. Shown are OD₅₅₀ values of solubilized crystal violet from a microtiter plate assay at 24 h for the strains listed. All strains are derivatives of V583, and the nomenclature for each response regulator insertion mutant corresponds to the designations assigned by Hancock and Perego (14). Error bars denote standard deviations.