Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis

Abstract

Enterococcus faecalis is a gram-positive commensal bacterium of the human intestinal tract. Its opportunistic pathogenicity has been enhanced by the acquisition of multiple antibiotic resistances, making the treatment of enterococcal infections an increasingly difficult problem. The extraordinary capacity of this organism to colonize and survive in a wide variety of ecological niches is attributable, at least in part, to signal transduction pathways mediated by two-component systems (TCS). Here, the ability of E. faecalis to utilize ethanolamine as the sole carbon source is shown to be dependent upon the RR-HK17 (EF1633-EF1632) TCS. Ethanolamine is an abundant compound in the human intestine, and thus, the ability of bacteria to utilize it as a source of carbon and nitrogen may provide an advantage for survival and colonization. Growth of E. faecalis in a synthetic medium with ethanolamine was abolished in the response regulator RR17 mutant strain. Transcription of the response regulator gene was induced by the presence of ethanolamine. Ethanolamine induced a 15-fold increase in the rate of autophosphorylation in vitro of the HK17 sensor histidine kinase, indicating that this is the ligand recognized by the sensor domain of the kinase. These results assign a role to the RR-HK17 TCS as coordinator of the enterococcal response to specific nutritional conditions existing at the site of bacterial invasion, the intestinal tract of an animal host.

FIG. 1.

eut operon for EA utilization in E. faecalis and Salmonella enterica serovar Typhimurium (S. typhimurium). (A) A diagram of the gene organization in the two operons is shown with the darker shade of gray representing genes that are present in one organism but missing in the other. The bar represents the extent of the fragment cloned in the pTCV-RR17p transcriptional fusion plasmid. (B) Table summarizing gene names, annotation numbers in genome databases, the results of ClustalW and BLAST analyses, and function. (C) Schematic representation of the basic EA metabolism in Salmonella. EA can enter the cell by diffusion either via EutH or by other, unknown routes (38). EutBC are the two subunits of the cobalamin-dependent EA ammonia-lyase. EutE is an acetaldehyde dehydrogenase, EutG is an alcohol dehydrogenase, and EutD is a phosphotransacetylase (6, 27, 39). Notably, the genes for the tricarboxylic acid cycle (TCA) are absent in the genome of E. faecalis V583.

FIG. 2.

Schematic representation of the structural and functional domains of the RR17 response regulator (190 amino acids) and the HK17 histidine kinase (477 amino acids). The numbers delineate the approximate initial and terminal amino acid residues for each domain, as deduced from the Pfam and FFAS03 database analyses.

FIG. 3.

Growth of E. faecalis on EA. ODs of E. faecalis cultures after 24 h of growth under aerobic (A) or anaerobic (B) conditions in M9HY medium containing 10 μg/ml of tetracycline. Gluc, M9HY medium plus 100 mM glucose; NCS, M9HY medium without carbon source; EA, M9HY medium plus 100 mM EA; EA + CoB12, M9HY medium plus 100 mM EA and 40 nM CoB12. The error bars represent standard deviations of three independent cultures.

FIG. 4.

Growth curves of E. faecalis parental strain V583T and RR17 mutant strain on EA as the sole carbon source in M9HY medium containing 10 μg/ml of tetracycline. EA was provided at 100 mM and CoB12 at 40 nM. Filled and open symbols represent growth under anaerobic and aerobic conditions, respectively. Squares represent E. faecalis V583T, while circles indicate the RR17 mutant. The error bars represent standard deviations calculated from two independent cultures. OD₅₂₅, OD at 525 nm.

FIG. 5.

Growth and survival of E. faecalis V583 under anaerobic conditions utilizing EA as a carbon source. CFU per ml was measured after 0, 24, 48, and 72 h. NCS, M9HY medium without carbon source; EA, 100 mM EA; CoB12, 40 nM CoB12; VitB12, 40 nM VitB12. The 48-h and 72-h time points of the EA plus CoB12 culture conditions are missing because counts were below the minimal experimental limit set for the experiment. The error bars denote standard deviations calculated from three independent cultures.

FIG. 6.

Transcription analysis of the RR17-encoding gene. Time course of β-galactosidase activity was taken from a culture of the parental strain, V583, carrying the pTCV-RR17p transcriptional lacZ fusion construct (A) or the pTCV-LacSpec vector (B). Cells were grown in M9HY medium containing 750 μg/ml spectinomycin and supplemented with the following: 100 mM EA plus 40 mM CoB12 (▪); 100 mM EA (•); NCS plus 40 nM CoB12 (○); and 100 mM glucose plus 40 nM CoB12 (▵).

FIG. 7.

EA stimulated HK17 autophosphorylation activity. (A) Concentration-dependent activation of the autophosphorylation activity of the HK17 histidine kinase (5 μM) was measured after 30 min of incubation in the presence of EA concentrations shown on the x axis. (B) Time course of HK17 (5 μM) autophosphorylation in the presence (▪) or absence (⧫) of EA (25 μM).