Molecular Characterization of Enterococcus faecalis N06-0364 with Low-Level Vancomycin Resistance Harboring a Novel d-Ala-d-Ser Gene Cluster, vanL

Abstract

Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 μg/ml, was found to harbor a novel d-Ala-d-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTm_L (membrane binding) and vanTr_L (racemase).

Among enterococci, there are currently three characterized d-Ala-d-Ser operons that confer low-level vancomycin resistance, vanC, vanE, and vanG (1, 5, 6, 10, 14). The vanC operon is intrinsic to Enterococcus gallinarum (vanC) and Enterococcus casseliflavus/flavescens (vanC2/3), while vanE and vanG have been acquired by a small number of Enterococcus faecalis strains (21). Resistance is achieved by essentially the same mechanism in the three types: the ligases, encoded by vanC, vanE, or vanG, catalyze the formation of d-Ala-d-Ser, which is incorporated into peptidoglycan precursors, which subsequently have a low binding affinity for vancomycin. The vanC and vanE operons are organized similarly, vanC or vanE, vanXY (d,d-dipeptidase-d,d-carboxypeptidase), vanT (serine racemase), vanR (response regulator), and vanS (sensor histidine kinase) (1, 5, 14). The vanG operon begins with a three-component regulatory system, vanU (transcriptional regulator), vanR, vanS, vanY (d,d-carboxypeptidase) in the vanG operon which is absent in the vanG2 operon, vanW (unknown function), and then vanG, vanXY, and vanT (6, 10). In this report we describe a novel d-Ala-d-Ser gene cluster, designated vanL.

The primers used in this study are listed in Table 1. PCR and thermal asymmetric interlaced PCR (TAIL-PCR) were carried out with AmpliTaq Gold (Applied Biosystems, Foster City, CA) or the FailSafe PCR system (Epicenter Biotechnologies, Madison, WI) as previously described (6, 7, 12, 22). DNA sequencing was carried out by the National Microbiology Laboratory's Genomics Core Facility. Homology comparisons were made using the BLAST suite of programs at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/BLAST/). Vancomycin induction was studied as previously described (19). E. faecalis JH2-2 was used as a recipient in filter mating experiments with selection for transconjugants on 50 μg/ml rifampin, 100 μg/ml fusidic acid, and 2 μg/ml vancomycin. Antimicrobial susceptibilities were determined using broth microdilution according to CLSI guidelines (9) and by Etest (AB Biodisk, Solna, Sweden).

TABLE 1.

Primers used in this study

Name	Sequence (5′ to 3′)a	Coordinates (bp)b	PCR no. (strand)c	Reference
vanA1	GGGAAAACGACAATTGC	NAd	NA	11
vanA2	GTACAATGCGGCCGTTA	NA	NA	11
vanB-F	AAGCTATGCAAGAAGCCATG	NA	NA	12
vanB-R	CCGACAATCAAATCATCCTC	NA	NA	12
ddlFm-1	ATTACAAAGGCAGAAAACCG	NA	NA	This study
ddlFm-2	TGTCAAAAAGAAATCGCACC	NA	NA	This study
ddlFs-1	TTATTTTGTTGTATGGCGGC	NA	NA	This study
ddlFs-2	AAAGTCAGTAAAACCAGGCA	NA	NA	This study
vanC1-93	GAAAGACAACAGGAAGACCGC	NA	NA	8
vanC2-93	ATCGCATCACAAGCACCAATC	NA	NA	8
vanD-U1	TATTGGAATCACAAATCCGG	NA	NA	7
vanD-U2	CGGCTGTGCTTCCTGATG	NA	NA	7
VANE1	TGTGGTATCGGAGCTGCAG	NA	NA	14
VANE2	GTCGATTCTCGCTAATCC	NA	NA	14
vanG5	TTCGATTTCATCAACTCTGC	NA	NA	This study
vanG6	CAGGAATACCTGTTGTTGG	NA	NA	This study
AD5	NTCGASTWTSGWGTT	NA	1 (+), 9 (−)	22
364U4	TTTCCGCCTAAATCAATTCC	1081-1100	1 (−)	This study
364U5	GAGTTTCTAGTACACTTACTG	1028-1048	1 (−)	This study
364U6	ATATTCCGAAGATTGACCTCC	988-1008	1 (−)	This study
AD3	AGWGNAGWANCAWAGG	NA	2 (+)	22
364U1	AGCATCCTCTAGCTTATCTGG	1537-1557	2 (−)	This study
364U2	TAGTGCGAATTATGAGCGTAG	1409-1429	2 (−)	This study
364U3	CACTCTGAGCAAACTCATGC	1371-1390	2 (−)	This study
V3	GARGATGGITSCATMCARGGW	1274-1293	3 (+)	15
V4	MGTRAAICCIGGCAKRGTRTT	1882-1902	3 (−)	15
364D4	AGCAAAGGTAAATATACCTGC	1728-1748	4 (+)	This study
vanXY-U	CCNACRTANCKRAARTGCCA	2468-2487	4 (−)	This study
vanXYL-D3	GCACATGATATCATAGCACC	2336-2355	5 (+)	This study
vanT-U	TDATNVDNGCNGGNARRTACCA	2941-2963	5 (−)	This study
vanTL-D1	TCTATCACTTTGGATATGTC	2869-2888	6 (+)	This study
vanT-U2	CCNARNCKRTGCAMNCCNGTRTC	3939-3961	6 (−)	This study
vanTL-D2	CTTATCCATTACGAACTTACC	3843-3873	7 (+)	This study
RserU1	AYRTCNGGNARCATNACRTC	4887-4906	7 (−)	This study
364RD1	AAGGGTTTCAAGTAACAACC	4804-4823	8 (+)	This study
SerU1	GGNGTNYKNARRTCRTGNGC	5863-5882	8 (−)	This study
364SD4	GAAGGGATATTGATTATCGGTGTG	5605-5628	9 (+)	This study
364SD5	CCAGAACAGCGTGTTAAGCTATC	5725-5747	9 (+)	This study
364SD1	TAATGCTGACAATCGCGCAG	5799-5818	9 (+)	This study
ISvL-D1	ATGGACTATTACGAGAGTACC	7481-7502	10 (+)	This study
ISvL-D2	TGATTCACAGGCTCCTTAGC	7524-7543	10 (+)	This study
ISvL-D3	TCAGAAGTTCAGTGAAGCTAC	7568-7588	10 (+)	This study
AD1	NGTCGASWGANAWGAA	NA	10 (−)	22

^aK is G or T, M is A or C, R is A or G, S is G or C, W is A or T, Y is C or T, D is A, G, or T, V is A, C, or G, I is inosine, and N is any base.

^bCoordinates are from accession no. EU250284.

^cPCR number as indicated in Fig. 1.

^dNA, not applicable.

E. faecalis N06-0364 (vancomycin MIC, 8 μg/ml) was isolated from a single patient after a screen for vancomycin-resistant enterococci was carried out 2 days after hospitalization for a total hip resection. No vancomycin-resistant enterococci were isolated from this patient or any other in the hospital on follow-up screening 7 days later. E. faecalis N06-0364 was susceptible to teicoplanin, ampicillin, fluoroquinolones, erythromycin, gentamicin, streptomycin, linezolid, and tetracyclines. PCR analysis was positive for ddl_{E. faecalis} but negative for ddl_{E. faecium}, vanA, and vanB in a multiplex PCR (12). PCRs for vanD, vanC, vanE, and vanG-type ligases were negative. A ∼600-bp product amplified with the V3/V4 degenerate primers for d-Ala-d-Xxx ligases (15) was sequenced, and its putative translation product exhibited 57% identity to the VanC ligase. Using DNA sequence alignments of homologous genes from existing d-Ala-d-Ser operons, a number of degenerate primers from conserved regions were designed (Table 1). These primers and gene-specific primers were used in PCRs to obtain a total region of ∼4 kb downstream of the V3/V4 PCR product (Fig. 1). An additional ∼1.2-kb region upstream and ∼2.5-kb region downstream were obtained by TAIL-PCR (Fig. 1). All products were sequenced to generate 8,295 bp of contiguous DNA sequence from E. faecalis N06-0364 (Fig. 1).

FIG. 1.

Schematic diagram of the VanL operon and flanking regions. Regions amplified and sequenced are indicated at the top and are numbered (see Table 1), with a triangle indicating a sequence-specific primer and an oval indicating a degenerate primer. The putative stem-loop region between vanS_L and ISEnfa364 is shown. The inverted repeats of ISEnfa364 are indicated by black bars, the two overlapping reading frames coding for tnpA are shown as arrows, and the in-frame stop codon is indicated by a vertical line. Coordinates are from GenBank accession number EU250284.

Analysis of the genes and their putative proteins indicated the presence of a novel d-Ala-d-Ser gene cluster, designated vanL. The percent identities of the vanL gene cluster proteins with the corresponding proteins from other d-Ala-d-Ser operons are shown in Table 2. The vanL gene cluster was organized essentially the same as the vanC and vanE operons; however, serine racemase activity appears to be encoded by two genes, vanTm_L and vanTr_L (discussed below). The putative VanL exhibited 51 and 49% identity to the VanE and VanC ligases, respectively, and the EKYQ motif involved in substrate recognition was conserved at residues 252 to 255 of VanL (Fig. 2A) (13).

TABLE 2.

Extents of identity of proteins from the VanL operon with the corresponding proteins from other D-Ala-D-Ser operons

Protein	% Identity with protein from other indicated operon
	VanL	VanXYL	VanTmLa	VanTrLa	VanRL	VanSL
VanC	49	46	45	51	72	52
VanE	51	41	44	53	59	39
VanG1	42	38	35	41	60	40
VanG2	42	38	34	40	62	40

^aAs aligned with the corresponding domain of VanT proteins.

FIG. 2.

Alignment of the VanL, VanE, and VanC proteins (A) and the VanXY_L and VanXY_C proteins (B). Identical residues are boxed in black. The EKYQ motif putatively involved in substrate binding is overlined by asterisks in panel A. The residues conserved in VanX, VanY, and VanXY proteins are indicated in panel B.

The putative VanXY_L exhibited 46% identity to VanXY_C and contained all the conserved active site residues of other VanXY proteins and also those found in VanX and VanY proteins (Fig. 2B) (20).

The third and fourth open reading frames, vanTm_L and vanTr_L, respectively, appear to encode serine racemase activity (Fig. 1). vanTm_L and vanTr_L are in different reading frames, and a single-base insertion between bases 3497 and 3564 (coordinates are from accession no. EU250284) would lead to a single 2,085-bp putative vanT_L gene. Whether extant vanT genes arose in this way or whether vanTm_L and vanTr_L evolved by a deletion in a vanT_C-like gene is purely speculative. An alignment of VanTm_L and VanTr_L with the VanT_C protein is shown in Fig. 3. VanTm_L exhibits 45% identity with the VanT_C N-terminal region, which is postulated to function in the transport of l-serine (2, 3). The VanTr_L protein exhibits 51% identity to the VanT_C C-terminal region and shares all the residues important in structure and function of pyridoxal 5′-phosphate-dependent alanine racemases (2) (Fig. 3). Since l-serine transport and racemase activities are not codependent for function (3), having these two domains separately encoded would not necessarily compromise VanT activity in E. faecalis N06-0364. The vanR_L and vanS_L genes follow vanTr_L (Fig. 1). Alignments of VanR_L and VanR_C and of VanS_L and VanS_C are shown in Fig. 4. The deduced VanR_L exhibited 72% identity to VanR_C and contained the conserved residues (D10, D53, and K102) found in response regulators from gram-positive two-component systems (Fig. 4A) (18). The deduced VanS_L exhibited 52% identity to VanS_C and contained the six conserved amino acid motifs (H, X, N, G1, F, and G2 boxes) of the C-terminal transmitter module of VanS proteins (16) (Fig. 4B). In addition, the VanS_L N-terminal region contained two transmembrane regions characteristic of the sensor proteins in two-component systems (Fig. 4B) (4). VanS_L has none of the substitutions (R200, D312, and G320) shown in VanS_C to be associated with constitutive vancomycin resistance (19). This is consistent with growth experiments which revealed that vancomycin resistance was inducible in E. faecalis N06-0364 (data not shown). Blast searches of the GenBank database with the ∼950 bp upstream of vanL failed to return any matches. Beginning 110 bp downstream of vanS_L is a 61-bp region with the potential to form a large stem-loop structure with a ΔG of −15.64 kcal/mol which may act as a transcriptional terminator (Fig. 1). Inserted 5 bp downstream of the putative stem-loop region is a 1,055-bp insertion sequence, designated ISEnfa364, with ends that are defined by 25-bp inverted repeats (IR) and that is most closely related to elements in the IS30 family (17). The ISEnfa364 transposase appears to be coded for by two overlapping reading frames whose products would presumably become linked through translational frameshifting (17). However, an in-frame stop codon would presumably lead to an inactive truncated protein. Typically, IS30 family members are flanked by short 2- to 4-bp direct repeats created upon insertion (17). ISEnfa364 is flanked by a single adenine residue. However, it was noted that the 4 bp flanking IR-L, ATGA, are repeated 2 bp downstream of IR-R.

FIG. 3.

Alignment of VanT_C with the VanTm_L and VanTr_L proteins. Identical residues are boxed in black, hydropobic transmembrane domains are indicated by solid lines, and the pyridoxal phosphate attachment site (PPAS) is labeled.

FIG. 4.

Alignments of the VanR_Land VanR_C proteins (A) and the VanS_L and VanS_C proteins (B). Identical residues are boxed in black. The conserved aspartate and lysine residues of gram-positive response regulators are labeled in panel A. The transmembrane regions are indicated by hatched lines, and the conserved amino acid motifs H, X, N, G1, F, and G2 of VanS proteins are labeled and indicated by solid lines in panel B.

The vanL region has a G+C content of 32%, while the E. faecalis genome has a G+C content of 38%, indicating acquisition from an organism with a more-AT-rich genome.

Despite several attempts, transfer of the vanL gene cluster could not be demonstrated in mating experiments with E. faecalis JH2-2 as a recipient. Plasmids were not visualized on gels from multiple plasmid preparations from E. faecalis N06-0364 (data not shown). It appears likely that the vanL gene cluster is located in the chromosome. All acquired d-Ala-d-Ser gene clusters identified to date, vanE, vanG, and vanG2, are chromosomally located (5, 6, 10, 14). Definitive proof of vanL functionality awaits cloning and transfer experiments and biochemical analysis of cell wall precursors.

vanL expands the so-called van alphabet and highlights the importance of characterization of E. faecalis and Enterococcus faecium isolates exhibiting low-level vancomycin resistance. As both the origin of the vanL gene cluster by this strain and the way this strain was acquired by this patient are unknown, the clinical significance of this finding remains to be established.

Nucleotide sequence accession number.

The vanL gene cluster characterized in this study has been assigned accession number EU250284 in the GenBank Database.