Competition between VanU Repressor and VanR Activator Leads to Rheostatic Control of Vancomycin Resistance Operon Expression

Summary

Various modes of gene regulation coexist in cells. One corresponds to the “switch on/ off” mechanism in which the regulator induces the promoter to a defined level. In another mechanism, the regulator activates the promoter to various levels according to the intensity or the nature of an input signal. In this study, we show that in VanG-type vancomycin resistant Enterococcus faecalis a repressor (VanUG) allows rheostatic expression of a target resistance promoter by competing with a response regulator (VanRG) which otherwise acts together with a sensor (VanSG) by a "switch on/off" mechanism as part of a two-component regulatory system. Unusually, both regulators are encoded in the same operon.

Introduction

Vancomycin-resistant enterococci are a major cause of nosocomial infections and an important public health problem because the treatment options for the infections they cause are very limited [1]. Vancomycin, which can be the only antibiotic effective against multiresistant clinical isolates, acts by binding to the C-terminal D-alanyl-D-alanine (D-Ala-D-Ala) residues of peptidoglycan precursors blocking the extracellular steps in peptidoglycan synthesis [2]. Resistance in Enterococcus is mediated by nine types of operons that produce modified peptidoglycan precursors ending in D-Ala-D-Lac (vanA, -B, -D, and-M) or D-Ala-D-Ser (vanC, -E, -G, -L, and-N) to which vancomycin bind with a low affinity and from the elimination of the high affinity precursors ending in D-Ala-D-Ala [3–6].

Expression of the vancomycin resistance operons is regulated by VanS/VanR-type two-component signal transduction systems composed of a membrane-bound histidine kinase (VanS-type) and a cytoplasmic response regulator (VanR-type) that acts as a transcriptional activator [3]. The sensors modulate the levels of phosphorylation of the regulators. In the presence of vancomycin, VanS acts primarily as a kinase that autophosphorylates and transfers its phosphate to VanR. Phosphorylated VanR binds to the promoters upstream from the vanRS regulatory and resistance operons leading to increased transcription of the regulatory and resistance genes [7–9]. The phosphatase activity of VanS-type sensors is required for negative regulation of the resistance genes in the absence of vancomycin preventing accumulation of VanR-type regulators phosphorylated by acetylphosphate or by kinases encoded by the host chromosome [7, 10].

VanG-type Enterococcusfaecalis clinical isolates from Australia and Canada are distinct from other Van-type enterococci. The chromosomal vanG cluster (Fig 1) confers resistance to vancomycin (MICs, 16 μg/ml) by inducible synthesis of precursors ending in D-Ala-D-Ser [11]. It contains the vanY_G,W_G,G,XY_G,T_G resistance genes, the last three strictly required for resistance encode, respectively, a VanG ligase to synthesize D-Ala-D-Ser, a VanXY_G D,D-carboxypeptidase to hydrolyse D-Ala-D-Ala, and a VanT_G membrane bound serine racemase to produce D-Ser (Fig 1). As opposed to the other van gene clusters, the vanG regulatory operon contains three genes, vanU_G, vanR_G, and vanS_G, encoding a "three component" regulatory system (Fig 1). Additional gene vanU_G encodes a transcriptional regulator belonging to the Xre protein family and of unknown function. The vanURS_G genes are co-transcribed, even in the absence of vancomycin, from the P_UG regulatory promoter, whereas transcription of the resistance genes is inducible and initiated from the P_YG resistance promoter [11].

Cryptic vanG-like operons are common in Clostridium difficile, a major human pathogen which is a target for vancomycin, and a vanU_G gene encoding a protein identical to VanU_G was found in a clinical isolate (GenBank N° AVLW01000050). A VanU_G-like protein (GenBank N° YP002939420), 79% identical with VanU_G, was detected in an Eubacterium associated with a two-component system controlling an ABC-type transporter and a protein (GenBank N°YP007781704) with 76% identity was reported in Ruminococcus bromii associated with a CheY related regulator and a partial vanG operon. These regulators have not been studied.

We report the role of VanU_G in the transcription of the vanG operon in E.faecalis. We show that VanU_G binds to the P_UG regulatory and P_YG resistance promoters and negatively regulates the vanURS_G regulatory and resistance operons. In contrast, VanR_G binds only to P_YG. It thus appears that, upon induction by vancomycin, the VanS_G sensor phosphorylates VanR_G which competes and displaces VanU_G from P_YG leading to transcription of the resistance operon in a dose dependent manner. Thus, rheostatic regulation of resistance gene expression results from binding of a repressor and an activator encoded in a single operon to the same promoter.

Results

VanU_G but not VanR_G binds to the P_UG regulatory promoter

Primer extension of the region upstream from vanU_G indicated that, irrespective of induction, the transcriptional start site for vanURS_G was located 22 bp upstream from the translation initiation codon of vanU_G [11]. The P_UG promoter consists of -35 and -10 regions corresponding to δ70 recognition sequences separated by 17 bp (Fig 2A). To determine if VanU_G and VanR_G bind to the P_UG regulatory promoter region and to identify putative specific binding sites, DNaseI footprinting experiments were carried out. A radiolabeled PCR probe corresponding to positions -247 to +110 relative to the transcription initiation site of P_UG was incubated with increasing amounts of purified VanU_G, VanR_G, and VanR_G phosphorylated (VanR_G-P) by acetyl phosphate. The P_UG region protected by VanU_G depended on the protein concentration, extending from -70 to -20 (positions relative to the transcription initiation site) overlapping the -35 sequence at a low concentration (Fig 2B, lane 6) and from -70 to +10 at higher concentrations (Fig 2B, lanes 7 and 8). The region (-70 to -20) contained two adjacent imperfect palindromic sequences likely corresponding to the binding motifs of VanU_G (Fig 2A). As opposed to the wild-type fragment, two DNA fragments containing double mutations in the imperfect dyad symmetry operator of P_UG were not retarded by VanU_G, indicating a key role in VanU_G binding (S1 Fig). The appearance of several DNase I hypersensitive sites (Fig 2B) corresponding to bending of the DNA duplex suggested binding of two VanU_G monomers or dimers. This is consistent with the presence of two inverted repeats in the P_UG region (Fig 2A) and with the two-step gel retardation (S1 Fig). In contrast to VanU_G, VanR_G and VanR_G-P did not bind to the P_UG promoter.

VanU_G acts as a repressor of the P_UG regulatory promoter

The vanG operon is part of a large genetic element and is transferable from E. faecalis BM4518 to E. faecalis JH2-2 from chromosome to chromosome [11]. Since clinical isolate BM4518 is not transformable, we studied the VanURS_G system in transconjugant BM4522 (JH2-2::vanG) (S1 Table). To determine the role of VanU_G on P_UG, the vanU_G, vanR_G, and vanS_G genes of BM4522 were inactivated individually by in-frame deletions leading to BM4720(ΔvanU_G), BM4721(ΔvanR_G), and BM4722(ΔvanS_G). Transcription of the regulatory genes was quantified by RT-qPCR. In BM4522, low level transcription occured at similar levels without and with various concentrations of vancomycin indicating that the P_UG promoter was not inducible by vancomycin (Fig 2C). In the absence of vanU_G, vanR_G and vanS_G were transcribed in the absence or presence of vancomycin at higher level (≈ 5-fold) from P_UG indicating that VanU_G acted as a repressor on this promoter region (Fig 2D). In the absence of vanR_G or vanS_G, transcription of the regulatory genes remained unchanged even in the presence of vancomycin.

To confirm regulation of P_UG by VanU_G, the vanURS_G genes were cloned into vancomycin susceptible Escherichia coli NR698 [12] under the control of promoter P_spank upstream from P_UG fused to a chloramphenicol acetyltransferase (CAT) reporter gene, the two promoters being separated by a transcription terminator (term) (Table 1). Subsequently, each of the three genes was inactivated. E. coli RNA polymerase bound to the P_UG promoter (S2A Fig) which was active in the new host, in the presence or in the absence of vancomycin (Table 1). CAT was produced at a maximum level in the absence of vanU_G by plasmids pAT952(P_spanktermP_UGcat), pAT966(P_spankvanR_GtermP_UGcat), and pAT969(P_spankvanRS_GtermP_UGcat) (Table 1). In contrast, in the presence of VanU_G, CAT production was decreased to similar basal levels by plasmids pAT965(P_spankvanU_GtermP_UGcat), pAT967(P_spankvanUR_GtermP_UGcat), and pAT968 (P_spankvanURS_GtermP_UGcat) (Table 1). These results confirmed that VanU_G acts as a strong repressor on the P_UG promoter.

The VanR_GS_G two-component system is functional

Transcription of the resistance genes is under the control of VanURS_G and, as discussed above, VanU_G negatively autoregulates vanURS_G transcription from the P_UG regulatory promoter. To determine if VanR_G and VanS_G acted as a two-component system and to study the putative interaction of VanU_G with these proteins, VanU_G, VanR_G, and the cytoplasmic histidine kinase domain of VanS_G were purified as C-terminal His-tag proteins (S1 Table). VanS_G autophosphorylated in the presence of [γ-³²P]-ATP (Fig 3A). When incubated with purified VanU_G or VanR_G, phosphorylated VanS_G transferred its phosphate group to VanR_G (Fig 3B) but not to VanU_G (Fig 3E). Phosphorylation of VanR_G was fast and efficient, occurring in less than a minute. To test the phosphatase activity of VanS_G, hydrolysis of VanR_G-P over time was analysed in the absence or in the presence of VanS_G. Purified [³²P]-VanR_G was stable in vitro for at least 30min and then dephosphorylated slowly (Fig 3C); addition of purified VanS_G increased dephosphorylation only slightly (Fig 3D–3G). These results indicate that VanRS_G was functional and had characteristics similar to those of other VanRS-type two-component systems [7, 9] and that VanU_G did not affect phosphorylation nor dephosphorylation of VanR_G and VanS_G (Fig 3E and 3F).

VanU_G and VanR_G bind to overlapping sites of the P_YG resistance promoter

To study the putative binding of VanU_G and VanR_G to the P_YG region and to identify specific binding sites, DNaseI footprinting experiments were carried out. The inducible P_YG promoter is composed of -35 (AAAACA) and -10 (TACAAT) regions separated by 16 bp which have similarity with δ70 recognition sequences, although the -35 sequence is not conserved consistent with the fact that the promoter is positively regulated (Fig 4B). Analysis of the P_YG region revealed three 12-bp directly repeated VanR_G binding motifs and a deduced consensus sequence (T/C)CGTANGAAA(T/A)T was analogous to that in the P_R and P_H vanA operon promoters [13]. In the P_UG region, similar sequences were not found (Fig 2A) which could explain lack of VanR_G binding. The radiolabeled probe corresponding to positions -163 to +69 relative to the transcription initiation point of the P_YG promoter and containing the three conserved sequences was incubated with increasing amounts of purified VanU_G, VanR_G, and VanR_G-P (Fig 4). The three proteins protected in a concentration-dependent manner an overlapping DNA region that included the three direct repeats. The P_YG region protected by VanU_G was much larger than that by VanR_G and VanR_G-P extending from -110 to -3 and overlapped the -35 sequence at 0.2 and 1μM (Fig 4A, lanes 17 and 18). The P_YG region protected by VanR_G and VanR_G-P extended from -100 to -56 at low concentration (Fig 4A, bracket I, lanes 3 and 8) and from -100 to -43 at higher concentrations (Fig 4A, bracket II, lanes 4 and 5, and 9 and 10). There were three binding motifs a, b, and c with different affinities for VanR_G and VanR_G-P in the P_YG promoter region (Fig 4). Only a slight difference in affinity in favor of VanR_G-P at 0.2μM was noted for the "a" site (Fig 4A, lane 2) compared with VanR_G which could be due to inefficient phosphorylation of VanR_G by acetylphosphate. VanR_G and VanR_G-P bound to the a and b sites (Fig 4A, lanes 2, 3, and 8) with higher affinity than to the c site (Fig 4A, lanes 4 and 5, and 9 and 10), whereas VanU_G bound to this DNA region with the same affinity (Fig 4A).

VanU_G allows rheostatic expression of the resistance genes

To study the consequences of the binding of VanU_G and VanR_G to overlapping regions of P_YG on the expression of the resistance genes, the VanT_G serine racemase was used as a reporter (Fig 5). In clinical isolate BM4518 and transconjugant BM4522, synthesis of the serine racemase was dependent on the concentration of vancomycin (Fig 5). In contrast, in BM4720(ΔvanU_G), the resistance operon was expressed at its maximum even at low concentrations of vancomycin. These results suggested that VanU_G acts as a repressor of P_YG and that, in its absence, there is no fine-tuning of resistance expression from this promoter. Thus, modulation of transcription by vancomycin was due to the phosphorylation level of VanR_G mediated by VanS_G provided that VanU_G was present. Surprisingly, as in the wild-type strain, induction was dependent on the concentration of the inducer in BM4721(ΔvanR_G) (Fig 5). This could be accounted for by the presence of a VanR homolog in the host. In fact, we found, in both E.faecalis BM4518 and transconjugant BM4522 which were entirely sequenced (GenBank N°PRJNA245745), a gene specifying a VanR'_G protein with 65% identity with VanR_G (S3A Fig). In BM4722(ΔvanS_G) there was no synthesis of VanT_G in the presence of vancomycin indicating that VanR_G and VanR'_G are not phosphorylated in the absence of VanS_G. Double mutant BM4723(ΔvanR_G, ΔvanR'_G) derived from E. faecalis BM4721(ΔvanR_G) was susceptible to vancomycin (MIC, 1μg/ml) and VanT_G production was no longer inducible by vancomycin, indicating cross-talk between VanS_G and VanR'_G (Fig 5). To avoid interference by this regulator, transcription from the P_YG promoter was studied in E.coli NR698 since E. coli RNA polymerase was able to bind to this promoter (S2B Fig). The vanURS_G, vanRS_G, and vanUS_G genes were cloned under the control of P_spank upstream from the P_YG transcriptionally fused to a cat gene generating pAT970 (P_spankvanURS_GtermP_YGcat), pAT971 (P_spankvanRS_GtermP_YGcat), and pAT972 (P_spankvanUS_GtermP_YGcat). In the absence of VanU_G, induction by vancomycin led to similar levels of CAT synthesis in the strain harboring pAT971 (P_spankvanRS_GtermP_YGcat) whatever the concentration of the inducer, whereas with pAT970 (P_spankvanURS_GtermP_YGcat) CAT production depended on the vancomycin concentration (Table 2). These results confirmed that, in the presence of vancomycin, VanU_G is required for rheostatic gene transcription from P_YG and that VanR_G phosphorylation is essential for expression of the resistance genes since, in the absence of this regulator in pAT972 (P_spankvanUS_GtermP_YGcat), the level of CAT activity was low, both without (74U±9) and with (104 U ± 13) vancomycin (0.30 μg/ml). In the absence of vancomycin, CAT activity was lower in E. coli producing vanU_G encoded by pAT970 (P_spankvanURS_GtermP_YGcat) than in its counterpart harboring pAT971 (P_spankvanRS_GtermP_YGcat). This confirms that VanU_G acts as a repressor on the P_YG resistance promoter (Table 2).

VanU_G and VanR_G compete for binding to the P_YG resistance promoter

Since VanU_G and VanR_G bound at overlapping sites of P_YG, to assess a possible effect of VanR_G on the binding of VanU_G, we performed DNaseI footprinting assays on the labeled P_YG probe with purified VanR_G and VanU_G (Fig 6). Low and medium concentrations (64 nM and 128 nM) of VanU_G which allow binding to P_YG were tested with increasing concentrations of VanR_G. Upon addition of VanR_G, the binding profile of VanU_G faded while that of VanR_G appeared and increased in a dose dependent manner (Fig 6A). In the reverse experiment two approriate concentrations of VanR_G were challenged by increasing concentrations of VanU_G and the binding of VanR_G decreased also in the presence of VanU_G (S4 Fig). In summary, VanU_G alone did not allow transcription of the resistance genes (Fig 6B). It thus appears that at a low concentration of vancomycin there was competition between VanU_G and VanR_G, the latter being partially phosphorylated, transcription of vanY_GW_GGXY_GT_G was low. In contrast, at high concentrations of vancomycin, VanR_G was efficiently phosphorylated and able to displace VanU_G leading to maximal transcription of the resistance genes from the P_YG promoter.

The presence of vanU_G reduces the fitness cost associated with expression of VanG-type resistance

To study the role of VanU_G in this sophisticated resistance mechanism, the fitness cost of BM4720(ΔvanU_G) compared with that of BM4522 in monocultures in the absence and in the presence of vancomycin (1 μg/ml) was analysed by determination of the growth rates (Table 3). The results showed that the growth rates of both strains were indistinguishable in the absence of vancomycin indicating that non induced VanG-type resistance is not costly for the host. In contrast, in the presence of vancomycin, the relative growth rate of BM4720(ΔvanU_G) (0.74) was significantly reduced when compared with that of BM4522 (0.93) indicating that increased expression of resistance was significantly more costly in the absence of vanU_G.

Discussion

Among the ubiquitous two-component regulators, VanR/VanS-type systems are one of the rare to control expression of genes mediating antibiotic resistance [3]. In the VanG-type strains, a membrane associated sensor kinase (VanS_G) which detects a signal associated with the presence of vancomycin in the environment and a cytoplasmic response regulator (VanR_G) that acts as a transcriptional activator are also present (Fig 1) and functional (Fig 3) but there is, in addition, a VanU_G transcriptional regulator (Fig 1).

In the two main VanA- and VanB-type systems, the regulatory genes (vanRS) and the resistance genes are transcribed from independent and coordinately regulated promoters, but VanR is the only known direct regulator of the resistance genes [3, 8, 13]. In VanG-type strains, co-transcription of vanURS_G is repressed from P_UG by VanU_G (Fig 2 and Table 1) and expression of the resistance genes from P_YG is activated by VanR_G and repressed by VanU_G (Fig 5 and Table 2). Thus, VanU_G regulates the resistance genes both directly, by binding to the P_YG promoter region (Fig 4), and indirectly by repressing synthesis of VanR_GS_G (Fig 5). Like other members of the XRE protein family (S3B Fig) [14–16], VanU_G binds to short repeated sequences which span the promoters (Fig 2A and 2B). Unlike the VanR and VanR_B proteins which bind to their own promoters [8, 13], VanR_G does not regulate its own expression (Fig 2). No sequences similar to the VanR_G consensus binding site are found in P_UG (Figs 2 and 4).

VanR_G, as VanR and VanR_B, belongs to the OmpR-PhoB subclass of response regulators that have the peculiarity to bind to their target promoters in the unphosphorylated or phosphorylated form [8, 13, 17, 18]. Phosphorylation of VanR and VanR_B enhances the affinity of the proteins for their respective regulatory P_R or P_RB and resistance P_H or P_YB promoter regions allowing increased transcription of the regulatory and resistance genes [8, 13]. In VanA-type strains, VanR and VanR-P bind to P_R and P_H regions which contain a single or two 12-bp conserved sites, respectively [13]. Comparison of the sequences of the P_UG and P_YG regions with the 12-bp consensus sequence spanned by VanR and VanR-P revealed three binding sites in the P_YG region with a consensus sequence (Fig 4B) similar to that in VanA-type resistance [13]. As for the regulatory P_R and resistance P_H promoters, the positioning of these sites in P_YG was upstream from the -35 motif. VanU_G, VanR_G, and VanR_G-P protected overlapping regions, the two latter binding to P_YG a and b sites with a higher affinity than to the c site (Fig 4). There are only two sites in the P_H promoter but VanR generated a more extensive footprint (80 bp for P_H) than VanR_G (42bp for P_YG) likely due to higher cooperativity of VanR. Although not essential for binding in vitro, phosphorylation of VanR_G increased its affinity for the P_YG resistance promoter (Fig 4). In the P_UG promoter region no sequences similar to the consensus were found (Fig 2A) which could explain the absence of binding of VanR_G and low-level transcription from the regulatory promoter.

In many instances, regulation of gene transcription in E.coli occurs essentially through control of the phosphatase activity of the sensor [19, 20]. In VanA- and VanB-type strains, the level of phosphorylation of VanR and VanR_B is modulated by the kinase and phosphatase activities of the VanS and VanS_B sensors [7, 10, 21]. Phosphatase activity is critical for response regulators, such as VanR and VanR_B, whose phosphorylated form is highly stable, to ensure that the protein is not permanently activated. In VanG-type strains, in the absence of VanU_G, induction by vancomycin led to maximal VanT_G serine racemase (Fig 5) or CAT synthesis (Table 2) even at low concentrations of the inducer. Since in the absence of VanU_G there was no modulation of resistance genes transcription from the P_YG promoter, this suggests that a low amount of VanR_G-P is sufficient to induce the resistance operon. VanU_G did not modulate VanR_G and VanS_G phosphorylation (Fig 4F) and was not phosphorylated by VanS_G (Fig 4E). Surprisingly, at least in vitro, the phosphatase activity of VanS_G was not very efficient (Fig 4D) in comparison with those of VanS or VanS_B [7, 9]. Expression of VanG-type resistance was thus inducible by vancomycin due to the presence of VanU_G as opposed to direct modulation of VanR activity by VanS in the other van operons. In the absence of vancomycin only VanU_G bound to the P_YG promoter; however when the concentration of vancomycin increased, VanR_G being more efficiently phosphorylated by VanS_G, displaced progressively VanU_G allowing gradual transcription of the resistance genes (Fig 6) as it is likely the case with VanR'_G, the VanR_G homolog encoded elsewhere in the chromosome. In B. subtilis, when both repressors SinR and SlrR are bound to the degU promoter, they can be displaced by the response regulator DegU leading to activation of the degU gene [22]. Also in B. subtilis, CcpC activates aconitase gene citB expression whereas CodY binds to its promoter and represses citB transcription [23]; PutR which is an activator essential for transcription of the putBCP operon for proline utilization is displaced by the CodY repressor [24].

VanU_G does not possess the characteristics of auxiliary regulators which can interact with histidine kinases, influencing signal perception and transduction. Nor does it interact with the response regulator to alter its phosphorylation status or its DNA binding ability, the recruitement of RNA polymerase on the promoter, or to sequester it through protein:protein interaction [25, 26]. The results presented here show that competition between the VanU_G repressor and the VanR_G activator for binding to the P_YG promoter may be responsible for the complex regulation of the resistance genes (Fig 6). This is an unusual example of rheostatic regulation of gene transcription due to binding competition between two regulators encoded in the same operon. It also elucidates an unsuspected strategy by which enterococcal clinical isolates regulate transcription of acquired genes for vancomycin resistance.

In previous work, we showed in VanB-type resistance that, despite the complex dual biochemical mechanism of resistance to vancomycin, its biological cost in enterococci is negligible when non induced, whereas a significant fitness reduction is observed when resistance is expressed in the presence of the inducer, the antibiotic itself [27]. Thus resistance is expressed exclusively when needed for bacterial survival. In VanG-type strains, tight regulation of resistance expression involves VanU_G which can thus be considered as a compensatory component, drastically reducing the biological cost associated with vancomycin resistance in the presence of antibiotic.

Materials and Methods

Bacterial strains, plasmids, and growth conditions

The origin and properties of the strains and plasmids are described in S1 Table. Escherichia coli TOP10 (Invitrogen, Groningen, The Netherlands) and NR698 (susceptible to vancomycin) [12] were used as a host for recombinant plasmids. E. coli BL21λDE3 [28], in which the T7 RNA polymerase gene is under the control of the inducible lacUV5 promoter carries the pREP4 plasmid allowing co-expression of the GroESL chaperonin to optimize recombinant protein solubility [29]. E. coli TG1 RepA [30] was used as a host for constructions in the pAT944(pGhost9Ωcat) vector (S1 Table). Kanamycin (50μg/mL) was used as a selective agent for cloning PCR products in the pCR-Blunt vector (Invitrogen). Ampicillin was used to select pUC1813 [31]. pDR111 (gift from David Rudner, Harvard University), which harbors the P_spank promoter between two fragments of the B.subtilis amyE gene, is a derivative of the P_spac-hy plasmid pJQ43 containing an additional lacO binding site to achieve a better repression in the absence of the IPTG inducer. P_spank is a lacI repressible IPTG inducible-promoter for gene overexpression. Spectinomycin (60μg/mL) and chloramphenicol (10μg/mL) were added to the medium to prevent loss of plasmids derived from pDR111(P_spank) and pAT944(pGhost9Ωcat), respectively. Enterococcus faecalis JH2-2 is a derivative of strain JH2 that is resistant to fusidic acid and rifampin [32]. In all experiments, strains were grown in brain heart infusion (BHI) at 37°C with shaking at 110 rpm.

Promoter DNA labeling

Labeled P_UG (357 bp) and P_YG (233 bp) fragments were generated by PCR with BM4518 total DNA as a template and primer pairs VanG12-VanG126 and VanSG6-YG10 (S2 Table), respectively, using a combination of an unlabeled primer with an end-labeled primer (625nM) with T4 polynucleotide kinase (0.075 U/μl) (New England Biolabs) and [γ³²P]-ATP (3000 Ci/mmol) (Perkin Elmer). The PCR reactions were carried out in a 50-μl volume and the products purified as described [8].

Gel shift assay

Purified labeled PCR products corresponding to wild-type and mutated P_UG promoter region fragments were recovered from a 6% polyacrylamide gel and used as a probe for the gel shift assay after addition of 100 μl of ammonium acetate (0.5 M) diluted in Tris buffer (10 mM, pH8.5) overnight at 37°C. The P_UG and mutated P_UG probes (10,000cpm each) were incubated with various concentrations of purified VanU_G regulator at 30°C for 20min in 20 μl of 50mM Tris-HCl (pH7.8) containing 20 mM MgCl2 and 0.1 mM dithiothreitol (DTT). After addition of the DNA dye solution (40% glycerol, 0.025% bromophenol blue and 0.025 xylene cyanol), the mixture was loaded on a 7.5% polyacrylamide gel in the absence of protein denaturants. The gels were dried and analysed by autoradiography.

DNase I footprinting

Complexes with the labeled promoter regions (5nM) were formed for 30 min at 30°C in 15 μl of buffer C (20 mM Hepes pH 8.0, 5 mM MgCl2, 50 mM potassium glutamate, 5 mM DTT, and 500μg/ml bovine serum albumin) using RNA polymerase of E. coli at 50 nM or VanU_G, VanR_G, or VanR_G-P at increasing concentrations. For DNase I experiments, 1.5 μl of DNase I solution (1 μg ml^-1 in 10 mM Tris-HCl, 10 mM MgCl2, 10 mM CaCl₂, 125 mM KCl) were added and incubated at 30°C for 10s when the labeled promoter regions were alone, or for 20 s when when RNA polymerase or VanU_G, VanR_G or VanR_G-P were present in the mixture. The reaction was stopped and all the samples were extracted, precipitated, washed, resuspended, and loaded on a sequencing gel as described [8]. Protected bands were identified by comparing the migration with that of the same fragment treated for the A+G sequencing reaction [33]. The gels were analysed by autoradiography.

Quantitative real-time RT-qPCR

Enterococci grown in 100 ml of brain heart infusion in 250-ml bottles, with and without vancomycin, at 37°C with shaking at 110 rpm to OD₆₀₀ = 0.8 were harvested. RNA was prepared using the Fast RNA ProBlue kit (MBP Biomedicals) according to the manufacturer's protocol, treated with DNase (Turbo DNA-free, Invitrogen), and checked for the absence of contaminant DNA in a standard PCR, using the same primers as for the RT-PCR. RNA concentrations were determined by measuring absorbance with a NanoDrop2000 (ThermoScientific). cDNA synthesis and RT-qPCR were performed with a Light Cycler RNA amplification kit SYBR greenI (Roche Diagnostic GmbH) in a total reaction volume of 19μl with 0.5 μM gene-specific primers (VanG129-VanG102 for vanU_G, VanRG2-VanRG10 for vanR_G, VanSG2-VanSG10 for vanS_G, and rpoB5-rpoB12 for rpoB) (S2 Table) according to the manufacturer's instructions. Amplification and detection of specific products were performed using the LightCycler sequence detection system (Roche) with the following cycle profile: 1cycle at 55°C for 20 min for the reverse transcription step, followed by 1 cycle at 95°C for 30 s, 45 cycles at 95°C for 5 s, 52°C for 15 s, and 72°C for 15 s. The level of every gene transcript was normalized relative to rpoB transcript levels.

Overproduction and purification of VanU_G, VanR_G, and VanS_G

Plasmids pAT940(pET28ΩvanU_G), pAT941(pET28ΩvanR_G), and pAT942(pET28ΩvanS_G) (S1 Table) were introduced into E. coli BL21λDE3/pREP4 [29]. The transformants were grown in 1 liter of LB medium in Fernbach flasks with shaking at 110 rpm at 28°C until OD600 = 0.8, IPTG (1 mM) was added to induce protein production, and incubation was pursued for 4 h. E.coli crude protein extracts were loaded on 1-ml His-Trap fast-flow columns (GE, Healthcare) equilibrated with buffer A (50mM NaH₂PO₄ pH 7.5, 300 mM NaCl, 30 mM imidazole) and the proteins were eluted with an imidazole gradient (30mM-500mM). Fractions were dialysed against buffer B (50mM NaH₂PO₄ pH 7.5, 300 mM NaCl, 50% glycerol). Protein concentration was determined using the Bio-Rad protein assay [34].

Autophosphorylation of VanS_G

Autophosphorylation of VanS_G (40 μg) was performed in a final volume of 100 μl of buffer A (final concentrations: 50 mM Tris-HCl, 50mM KCl and 1 mM MgCl2, pH7.5). The reaction was initiated by the addition of 5 μl of ATP (1mM final) containing 200 μCi of [γ-32P]ATP and incubated at room temperature for 1 h. ATP was removed using 500 μl Sephadex G-50 spin column equilibrated with buffer A. The reaction was quenched by the addition of 5 μl of β-mercaptoethanol-stop solution (Sigma), followed by electrophoresis on 12% NuPAGE Novex Bis-Tris gels (Invitrogen) in MOPS buffer (1X), and autoradiography.

Phosphorylation of VanU_G and VanR_G by VanS_G

Phosphotransfer to purified VanU_G and VanR_G were carried out in buffer A. The reaction was initiated by the addition of 10 μl of the purified autophosphorylation reaction mixture of VanS_G (40 μg) described above to a 15 μl reaction mixture containing VanU_G or VanR_G (55 μg each). After incubation for various periods of times at room temperature, the phosphotransfer reactions were quenched by the addition of stop solution (Sigma) followed by electrophoresis on 12% NuPAGE Novex Bis-Tris gels (Invitrogen) in MOPS buffer (1X) and autoradiography.

Phosphorylation of VanU_G and VanR_G by acetyl[³²P]phosphate

VanU_G (220 μg) or VanR_G (225 μg) were incubated in 100 μl of buffer B (50 mM Tris-HCl, pH7.8, 20 mM MgCl2, 0.1 mM dithiothreitol) containing 178 pmol (3.3 μCi) of acetyl[³²P]phosphate (Hartmann Analytical, Germany) at room temperature for 60 min. Excess acetyl[³²P]phosphate was removed using Sephadex G-50 spin columns equilibrated with buffer B. Aliquots (10 μl) were withdrawn at designated time points, and the phosphorylation reactions were quenched with β-mercaptoethanol-stop solution followed by electrophoresis on 15% SDS-polyacrylamide gels and autoradiography.

Hydrolysis of phospho-VanU_G and phospho-VanR_G by VanS_G

The VanU_G (220 μg) and VanR_G (225 μg) response regulators were labelled with acetyl[³²P]phosphate for 1 h at room temperature as described above, and 52 μg of VanS_G histidine kinase were added, and incubation was pursued for various periods of times. Aliquots (10 μl) were withdrawn at designated time points and the reactions were stopped, followed by electrophoresis on 15% SDS-polyacrylamide gels and autoradiography.

Plasmid construction

The plasmids were constructed as follows.

Construction of pAT940, pAT941 and pAT942. pAT940(pET28ΩvanU_G) and pAT941(pET28ΩvanR_G)

A 225-bp BsaI-XhoI fragment corresponding to the vanU_G coding sequence amplified with UG1 and UG2 (S2 Table) and a 705-bp BsaI-XhoI fragment corresponding to the vanR_G coding sequence amplified by using oligonucleotides RG1 and RG2 (S2 Table) and BM4518 [11] total DNA as a template, were cloned in the NcoI and XhoI sites of modified pET28 [35] to generate plasmids pAT940(pET28ΩvanU_G) and pAT941(pET28ΩvanR_G). Oligodeoxynucleotide UG1 contained a BsaI restriction site designed to generate a cohesive end compatible with NcoI and 16 bases complementary to codons 1–6 of vanU_G of BM4518 (S2 Table). Oligodeoxynucleotide UG2 contained a XhoI site replacing the TGA stop codon and 21 bases complementary to codons 69–75 of vanU_G. Oligodeoxynucleotide RG1 contained a BsaI restriction site designed to generate a cohesive end compatible with NcoI and 16 bases complementary to codons 1–6 of vanR_G of BM4518. Oligodeoxynucleotide RG2 contained a XhoI site replacing the TGA stop codon and 21 bases complementary to codons 229–235 of vanR_G.

pAT942(pET28ΩvanS_G)

A cytoplasmic portion of the vanS_G gene of strain BM4518 was amplified using BM4518 total DNA as a template and primer pair SG1-SG3 (S2 Table). Oligodeoxynucleotide SG1 contained a BsaI restriction site designed to generate a cohesive end compatible with NcoI, and 16 bases complementary to codons 88–93 of vanS_G. Oligodeoxynucleotide SG3 contained a XhoI site in place of the TAG stop codon and 21 bases complementary to codons 361–367 of vanS_G. The 842-bp pCR product from vanS_G was digested by BsaI and XhoI and cloned between the NcoI and XhoI restriction sites of plasmid pET28 to generate plasmid pAT942(pET28ΩvanS_G).

Construction of pAT944(pGhost9Ωcat)

The XbaI cassette containing the chloramphenicol acetyltransferase cat gene with its own promoter was amplified from DNA of plasmid pAT943(pUC1318ΩPcat) with primers pG9CAT_NH2 and pG9CAT_COOH (S2 Table) which contain a XbaI restriction site allowing the replacement of the XbaI fragment containing the erythromycin resistance gene in pGhost9 [36] to generate plasmid pAT944(pGhost9Ωcat).

Construction of pAT945(pGhost9CmΩΔvanU_G), pAT946(pGhost9CmΩΔvanR_G), pAT947(pGhost9CmΩΔvanS_G,), and pAT973(pGhost9CmΩΔvanR'_G)

The vanU_G, vanR_G, and vanS_G genes of the vanG operon and vanR'_G from BM4518 were inactivated by deletion using splicing-by-overlap extension PCR in two steps and cloned into the thermosensitive shuttle plasmid pAT944(pGhost9Ωcat) using XhoI and PstI restriction sites to generate plasmids pAT945(pGhost9CmΩΔvanU_G), pAT946(pGhost9CmΩΔvanR_G), pAT947(pGhost9CmΩΔvanS_G), and pAT973(pGhost9CmΩΔvanR'_G). The primers used for the construction of the deletant alleles and the extent of the deletions are reported in S2 Table. A SmaI restriction site was added in the primers to screen for integration in the corresponding chromosomal gene. Briefly, the remnants of the vanU_G, vanR_G, vanS_G and vanR'_G genes of BM4518 were first amplified from total DNA of BM4518 as a template using primers UG3-UG4 and UG5-UG6 for ΔvanU_G, UG3-RG4 and RG5-RG7 for ΔvanR_G, SG4-SG5 and SG6-SG7 for ΔvanS_G, RG10-RG11 and RG12-RG13 for ΔvanR'_G and, in a second step, the resulting PCR products were amplified with UG3 plus UG6, UG3 plus RG7, SG4 plus SG7, and RG10 plus RG13 respectively, to obtain ΔvanU_G, ΔvanR_G, ΔvanS_G and ΔvanR'_G.

Construction of pAT949 and derivatives

Plasmid pAT949(pDR111ΩP_spankcat) was constructed by cloning the HindIII-SphI fragment of pAT948(pUC1813Ωcat) carrying the cat cassette in pDR111(P_spank) digested with the same enzymes allowing a directional cloning of the cat reporter gene under the control of the inducible P_spank promoter.

pAT950 (pDR111ΩP_spanktermcat)

A 66-bp HindIII-SalI fragment corresponding to the transcription terminator of gene 32 from bacteriophage T4 [37] was amplified by PCR with oligodeoxynucleotides T4F-HindIII and T4R-SalI/NheI (S2 Table). Primer T4F-HindIII contained HindIII and NheI restriction sites. Primer T4R-SalI/NheI contained SalI and NheI restriction sites. The HindIII and SalI restriction sites allowed directional cloning of the transcription terminator (term) from bacteriophage T4 under the control of the inducible P_spank promoter and upstream from the cat reporter gene of the pAT949(pDR111ΩP_spankcat) shuttle vector.

pAT951(pDR111ΩP_spankvanU_Gcat)

The vanU_G gene of BM4518 was amplified using primer pair UG_NH2 and UG_COOH (S2 Table) and total DNA of the corresponding strain as a template. Oligodeoxynucleotide UG_NH2 contained BsaI and HindIII restriction sites, a RBS, and 6 bases complementary to vanU_G including the ATG (translation initiation) codon. Oligodeoxynucleotide UG_COOH harbored SalI and NheI restriction sites, the stop codon, and 15 bases complementary to the 3’ end sequence of vanU_G from BM4518. The BsaI and SalI restriction sites allowed directional cloning of a 249-bp fragment of vanU_G downstream from the inducible P_spank promoter and upstream from the cat gene of the pAT949(pDR111ΩP_spankcat) shuttle vector to generate pAT951(pDR111ΩP_spankvanU_Gcat).

pAT952(pDR111ΩP_spanktermP_UGcat) and pAT953(pDR111ΩP_spankvanU_GP_UGcat)

The regulatory P_UG (183 bp) promoter was amplified by PCR from BM4518 total DNA with oligodeoxynucleotides PUG1 and PUG2 (S2 Table). Primers PUG1 and PUG2 contained a NheI and a SalI restriction site, respectively, which allowed directional cloning of P_UG upstream from the cat gene of pAT950(pDR111ΩP_spanktermcat) to generate pAT952(pDR111ΩP_spanktermP_UGcat) or allowed directional cloning of P_UG downstream from vanU_G and upstream from the cat reporter gene of pAT951(pDR111ΩP_spankvanU_Gcat) to generate pAT953(pDR111ΩP_spankvanU_GP_UGcat).

pAT954(pDR111ΩP_spankvanR_GP_UGcat)

A 754-bp HindIII-NheI fragment corresponding to the vanR_G coding sequence with its RBS, initiation and stop codons was amplified by PCR from BM4518 with oligodeoxynucleotides RG_NH2 and RG_COOH (S2 Table). Primer RG_NH2 contained a HindIII restriction site. Primer RG_COOH comprised SalI and NheI restriction sites, the stop codon, and 14 bases complementary to the 3’ end of vanR_G from BM4518. The HindIII and NheI restriction sites allowed directional cloning of the vanR_G gene under the control of the inducible P_spank promoter and upstream from P_UG and the cat gene of pAT952(pDR111ΩP_spanktermP_UGcat).

pAT956(pDR111ΩP_spankvanUR_GP_UGcat), pAT958(pDR111ΩP_spankvanRS_GP_UGcat), pAT960(pDR111ΩP_spankvanURS_GP_UGcat) pAT961(pDR111ΩP_spankvanRS_GP_YGcat)and pAT962(pDR111ΩP_spankvanURS_GP_YGcat)

The vanUR_G, vanRS_G, and vanURS_G genes of BM4518 were amplified using primer pairs UG_NH2-RG_COOH, RG_NH2-SG_COOH, and UG_NH2-SG_COOH (S2 Table), respectively, and BM4518 total DNA as a template. Oligodeoxynucleotides UG_NH2 and RG_NH2 harbored a HindIII restriction site and 21 bases complementary to the sequence upstream from vanU_G or 17 bases complementary to the sequence upstream from vanR_G. Primers RG_COOH and SG_COOH contained each SalI and NheI restriction sites, the stop codon and 14 or 13 bases complementary to the 3' end of respectively vanR_G and vanS_G of BM4518. The HindIII and SalI restriction sites allowed directional cloning of vanUR_G, vanRS_G, and vanURS_G upstream from the cat reporter gene of shuttle vector pAT949(pDR111ΩP_spankcat) carrying the inducible P_spank promoter to generate pAT955(pDR111ΩP_spankvanUR_Gcat), pAT957(pDR111ΩP_spankvanRS_Gcat), and pAT959(pDR111ΩP_spankvanURS_Gcat). The 183-bp NheI-SalI fragment carrying the P_UG promoter obtained above by amplification was cloned in pAT955(pDR111ΩP_spankvanUR_Gcat), pAT957(pDR111ΩP_spankvanRS_Gcat), and pAT959(pDR111ΩP_spankvanURS_Gcat) digested with the same enzymes to generate pAT956(pDR111ΩP_spankvanUR_GP_UGcat), pAT958(pDR111ΩP_spankvanRS_GP_UGcat), and pAT960(pDR111ΩP_spankvanURS_GP_UGcat). The 177-bp NheI-SalI fragment carrying the P_YG resistance promoter amplified by PCR from BM4518 DNA with primers PYG1 and PYG2 (S2 Table) was cloned in pAT957(pDR111ΩP_spankvanRS_Gcat), and pAT959(pDR111ΩP_spankvanURS_Gcat) digested with the same enzymes to generate, respectively, pAT961(pDR111ΩP_spankvanRS_GP_YGcat)and pAT962(pDR111ΩP_spankvanURS_GP_YGcat).

pAT964(pDR111ΩP_spankvanU_Gtermcat), pAT965(pDR111ΩP_spankvanU_GtermP_UGcat), pAT966(pDR111ΩP_spankvanR_GtermP_UGcat), pAT967(pDR111ΩP_spankvanUR_GtermP_UGcat), pAT968(pDR111ΩP_spankvanURS_GtermP_UGcat), pAT969(pDR111ΩP_spankvanRS_GtermP_UGcat), pAT970(pDR111ΩP_spankvanURS_GtermP_YGcat), and pAT971 (pDR111ΩP_spankvanRS_GtermP_YGcat)

The NheI terminator fragment amplified by PCR with oligodeoxynucleotides T4F-NheI and T4R-NheI/KpnI (S2 Table) was cloned, respectively, in pAT951(pDR111ΩP_spankvanU_Gcat), pAT953(pDR111ΩP_spankvanU_GP_UGcat), pAT954(pDR111ΩP_spankvanR_GP_UGcat), pAT956(pDR111ΩP_spankvanUR_GP_UGcat), pAT960(pDR111ΩP_spankvanURS_GP_UGcat), pAT958(pDR111ΩP_spankvanRS_GP_UGcat), pAT962(pDR111ΩP_spankvanURS_GP_YGcat) and pAT961(pDR111ΩP_spankvanRS_GP_YGcat) digested with NheI.

pAT972(pDR111ΩP_spankvanUS_Gtermcat)

The 1,144-bp fragment containing the vanS_G gene of BM4518 was amplified using primer pair SG_NH2-SG_COOH (S2 Table) and total DNA of the corresponding strain as a template. The NheI and SalI restriction sites allowed directional cloning of vanS_G downstream from the vanU_G gene and upstream from the cat gene of pAT951(pDR111ΩP_spankvanU_Gcat) to generate pAT963(pDR111ΩP_spankvanUS_Gcat).

The EcoRI fragment harboring the vanUS_G' genes from pAT963(pDR111ΩP_spankvanUS_Gcat) was replaced by the EcoRI fragment carrying the vanRS_G' genes of pAT971(pDR111ΩP_spankvanRS_GtermP_YGcat) to generate pAT972(pDR111ΩP_spankvanUS_Gtermcat).

Construction of strains

Plasmids pDR111, pAT949, pAT950, pAT952, pAT964, pAT965, pAT966, pAT967, pAT968, pAT969, pAT970, pAT971, and pAT972 were introduced by transformation into vancomycin susceptible E. coli NR698 and transformants were selected on agar containing chloramphenicol (10 g/ml) or ampicillin (100 μg/ml, for pDR111) (Tables 1 and 2).

In Gram-positive bacteria, pGhost9 [36] which replicates at 28°C but is lost above 37°C, allowed construction of E.faecalis BM4522 derivatives by insertional inactivation. Plasmids pAT945(pGhost9CmΩΔvanU_G), pAT946(pGhost9CmΩΔvanR_G), and pAT947(pGhost9CmΩΔvanS_G) were electrotransformed into E. faecalis BM4522 [11] to generate, respectively, BM4720(ΔvanU_G), BM4721(ΔvanR_G), and BM4722(ΔvanS_G) (S1 Table). Plasmid pAT973(pGhost9CmΩΔvanR'_G) was electrotransformed into E. faecalis BM4721(ΔvanR_G) to generate the double mutant BM4723(ΔvanR_G, ΔvanR'_G). Transformants were selected at the permissive temperature (28°C) on M17 plates containing 10g/ml of chloramphenicol and 0.5% glucose. A colony of each transformant was inoculated into 50 ml of M17 broth containing 0.5% glucose and incubated for 2h at 28°C. The culture was then shifted to a non-permissive temperature (42°C) for 2 h and integrants, following a first recombination event, were selected at 42°C on M17 agar containing chloramphenicol (10g/ml). Plasmid excision, by a second recombination event, was favored by subculturing at 28°C in the absence of chloramphenicol and plasmid loss was screened for by plating at 42°C on M17-glucose followed by replica plating on chloramphenicol. The integration locus was determined by PCR following digestion with SmaI and sequencing.

Enzyme assays

For preparation of extracts, 8 ml of an overnight culture were added to 100 ml of broth in the absence or in the presence of vancomycin and strains were grown until OD₆₀₀ = 0.8 in 250 ml bottles with shaking at 110 rpm. The cells were harvested by centrifugation, washed in 0.1M phosphate buffer pH 7.0, resuspended in the same buffer, lysed by sonication, followed by centrifugation at 10,000 g during 45 min. The resuspended pellet for VanT_G racemase [11] and supernatant for CAT activity, were assayed as described [38].

Genome sequencing, assemblies and annotation

Total DNA from BM4518 and BM4522 strains was purified and sequencing library preparation was carried out using the Nextera DNA Sample Preparation kit (Illumina, San Diego, CA), according to manufacturer’s specifications. Quality and quantity of each sample library was measured on an Agilent Technologies 2100 Bioanalyzer (Santa Clara, CA). Libraries were normalized to 2nM, multiplexed and subjected to 250-bp paired end sequencing (Illumina MiSeq). On average, 5 million high-quality paired-end reads were collected for each strain, representing >220-fold coverage of the ~2.9 Mb genomes. Reads were assembled de novo utilizing CLC Genomics Workbench (CLC bio, Cambridge, MA). Functional annotations were performed using a custom pipeline as described previously [39].

Determination of growth rates

Growth rates were determined in microplates coupled to a spectrophotometer iEMS reader (Labsystems). Strains were grown overnight at 37°C without or with 1 μg/ml of vancomycin. The cultures were diluted at OD 0.15 into 10 ml of broth without or with vancomycin (1μg/ml) and grown at 37°C with shaking until the beginning of the stationary phase. The cultures were diluted 1/1,000 to inoculate 10⁵ bacteria into 200 μl of broth in a 96-well microplate that was incubated overnight at 37°C with shaking. Absorbance was measured at 600 nm every 3 min. Each culture was replicated three times in the same microplate. Growth rates performed in three independent experiments were determined at the beginning of the exponential phase and the relative growth rates were calculated as the ratio of the growth rate of the strain induced by vancomycin versus that of the non induced strain.

Supporting Information

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