Both members of each two-component system identified in the E. faecalis genome were queried against protein and nucleotide sequence databases by using the BLAST programs. Chromosomal regions immediately surrounding the kinase and response regulator genes were also analyzed, as some two-component systems are adjacent to genes that they control or are involved in their function.
The most significant matches observed are summarized in Tables and . The percentages of identity and similarity reported indicate that, among the 17 two-component systems identified in E. faecalis, two have been functionally characterized (VanSBRB and FsrCA) and four are most likely orthologues of functionally characterized systems (HK-RR02, HK-RR04, HK-RR07, and HK-RR08). For the remaining systems, notwithstanding the high level of similarity reported, no function assignment can be done at this time.
The two-component system HK-RR01 shares sequence similarity with YesM-YesN from B. subtilis
) as well as a similar two-component pair from E. faecium
). Apart from the unusual structural properties previously discussed, no biological function can be inferred for this system.
The HK-RR02 two-component system shares similarity with the LytSR system in Staphylococcus aureus
and the LytST system in B. subtilis
. The S. aureus
LytSR system was first identified by Brunskill and Bayles (7
) as a regulatory locus affecting the rate of autolysis and penicillin tolerance. Subsequent work has identified the genes regulated by the LytSR system as a bicistronic operon, lrgAB
, whose gene products modulate murein hydrolase activity and penicillin tolerance (27
). For B. subtilis
, a comprehensive analysis of the two-component regulatory systems by use of DNA microarrays has revealed that LytST controls the expression of a bicistronic operon, designated ysbAB
). The YsbB protein is highly related to the lrgB
gene product. Downstream of the E. faecalis
HK-RR02 system is an operon with genes that share strong similarity to those of the lrgAB
operons, suggesting the possibility that this two-component system is the E. faecalis
orthologue of the Lyt system of S. aureus
and B. subtilis
The HK-RR03 two-component system shares extensive sequence similarity with the B. subtilis
YvqEC two-component system and a recently identified homologue in S. aureus
, designated VraSR (39
). In S. aureus
, the overexpression of the response regulator VraR leads to an increased resistance to vancomycin. What this system senses for the cell and how its expression affects vancomycin resistance in S. aureus
remain to be elucidated. Another interesting feature of this two-component system is that it is highly conserved in most gram-positive pathogenic bacteria, including S. aureus
, S. pyogenes
, and S. pneumoniae
, with an amino acid identity of nearly 60 to 70% and a homology of 80 to 90% within the catalytic domain of the kinases and the response regulator sequences (17
Of the 10 members of the group IIIA kinase-OmpR regulator family in E. faecalis, four could be assigned a putative functional role based on strong sequence similarity to proteins with known function.
HK-RR04 shares strong similarity with the PhoPR system of B. subtilis
(54% amino acid identity with PhoP and 30% amino acid identity with PhoR) (32
). The lower level of identity with the PhoR sensor kinase is due to the fact that no similarity exists between the amino-terminal ends of these two kinases. This may indicate that sensing modules for these proteins may have evolved independently.
The histidine kinase of the HK-RR07 system shares 45% amino acid identity and 70% similarity with the B. subtilis
YycG kinase. This kinase, along with its cognate response regulator YycF, is highly conserved in gram-positive bacteria (4
). It has been established that this two-component system is essential in B. subtilis
), S. aureus
), and S. pneumoniae
). Consistent with these findings, the yycF
gene has also been found to be essential in E. faecalis
(L. Hancock and M. Perego, unpublished observation). A recent report has linked the YycFG system in B. subtilis
to the regulation of the ftsAZ
), which may, in part, explain the essential nature of this system. The nature of the signal to which YycG might respond remains a mystery, but some clues may be derived from the fact that it possesses a PAS domain (29
). PAS domains are known to bind small regulatory molecules such as NAD, heme, flavin adenine dinucleotide, and, as reported more recently, ATP (3
HK-RR08 is homologous to a recently described two-component system of S. pneumoniae
, designated VncRS for its role in vancomycin tolerance (35% identity and 56% similarity between kinases; 62% identity and 79% similarity between response regulators) (53
). Similarity to the VncRS system extends to the three open reading frames upstream of HK-RR08, which also seem to encode a putative ABC transport system (52
). Between the two-component system and the ABC transporter, we identified a small open reading frame encoding a putative peptide of 43 amino acids. This is reminiscent of the pep27
gene upstream of VncRS, which encodes a 27-amino-acid peptide. This peptide has recently been shown to induce multiple cell death mechanisms, including antibiotic-induced autolysis, through modulation of the activity of the VncRS system (52
HK-RR11 is 100% identical to both the histidine kinase and the response regulator of the VanB-type vancomycin resistance determinant. A comprehensive review of this system has recently been reported (8
). This level of homology suggests that HK-RR11 was acquired by horizontal gene transfer (vancomycin resistance transposon).
For the remaining seven systems of group IIIA, two, HK-RR10 and HK-RR12, have similarities to known systems, but making a functional correlation is more hazardous.
The HK-RR10 system shows strong similarity to the LisRK system of Listeria monocytogenes
) and the CsrRS system of S. pyogenes
). HK-RR10 has recently been implicated in the stress response and virulence of E. faecalis
and was renamed EtaSR (62
HK-RR12 is homologous to the KdpDE two-component system from both gram-negative and gram-positive organisms (66
). An unusual feature of this system is that, unlike other members of the class IIIA kinase-OmpR regulator systems, whose genes are arranged in the regulator-to-kinase order, it has the kinase-coding gene followed by the response regulator-coding gene. The kinase itself is unusual for the class IIIA family of sensor histidine kinases in that it possesses four predicted transmembrane domains; the other members of this family possess only two.
With regard to the four remaining members of the class IIIA kinase-OmpR regulator systems in E. faecalis (HK-RR05, HK-RR06, HK-RR09, and HK-RR13), HK-RR06 has homology to the B. subtilis YclKJ system, whose function is unknown; the other three members appear to be unique to this organism since no significant similarities were identified in other bacteria, with the exception that strong homologues to HK-RR06 and HK-RR13 were identified in the preliminary sequencing data from the E. faecium genome project. RR05 and RR13 actually show some similarities to known response regulators (Table ), but the HK05 and HK13 proteins do not show sufficient similarities to the corresponding histidine kinase to justify any speculation on functional relationship.
According to Fabret et al. (14
), HK-RR14 of E. faecalis
strain V583 has a histidine kinase belonging to the class IV kinase family. Apart from the prediction that this kinase contains a single transmembrane domain, no other relevant structural features were evident and little sequence similarity with other two-component systems was found (Tables and ).
HK-RR15 corresponds to the recently described Fsr system of E. faecalis
). This system has been shown to regulate the synthesis of two secreted proteins, gelatinase and a serine protease. Furthermore, in an intraperitoneal infection model, mutants with disruptions in the kinase gene fsrC
or the response regulator gene fsrA
are attenuated in the virulence of E. faecalis
The kinase of the HK-RR16 system is predicted to contain seven transmembrane domains, and the region surrounding its phosphorylated histidine is similar to the one in FsrC. The RR16 response regulator is characterized by a DNA-binding domain that likely belongs to the LytTR (“litter”) family of transcriptional regulators (49
). The E. faecalis
RR02 and RR15 response regulators also belong to this family. Thus, this system may belong to the Agr kinase family (49
). Apart from predicted structural findings, however, no functional data are available concerning the role of this system in the biology of E. faecalis
The HK-RR17 system is the only one in E. faecalis with a cytoplasmic histidine kinase, but it does not have any significant similarity to known two-component systems.
Not surprisingly for a nonmotile organism, E. faecalis is devoid of a two-component system for chemotaxis and motility.