©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cross-talk between the Histidine Protein Kinase VanS and the Response Regulator PhoB
CHARACTERIZATION AND IDENTIFICATION OF A VanS DOMAIN THAT INHIBITS ACTIVATION OF PhoB (*)

(Received for publication, March 10, 1995; and in revised form, July 14, 1995)

Stewart L. Fisher (1)(§) Weihong Jiang (2) Barry L. Wanner (2) Christopher T. Walsh (1)(¶)

From the  (1)Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 and the (2)Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

VanS is a two-component transmembrane sensory kinase that, together with its response regulator VanR, activates the expression of genes responsible for vancomycin resistance in Enterococcus faecium BM4147. In this report, we demonstrate that the cytoplasmic domain of VanS (including residues Met to Ser) is capable of high level activation (>500 fold) of the Escherichia coli response regulator PhoB in vivo in the absence of its signaling kinases PhoR, CreC (PhoM), or acetyl phosphate synthesis. In vitro experiments carried out on the purified proteins confirmed that the activation is due to efficient cross-talk between VanS and PhoB, since phospho-VanS catalyzed transfer of its phosphoryl group to PhoB with approx90% transfer in 5 min at a 1:4 VanS/PhoB stoichiometry. However, the rate of transfer was at least 100-fold slower than that observed between phospho-VanS and VanR. The in vivo activation of PhoB was used as a reporter system to identify peptide fragments of VanS capable of interfering with activation by VanS(Met-Ser), in order to identify an interaction domain. A library of plasmids encoding fragments of VanS(Met-Ser) was constructed using transposon mutagenesis, and a subpopulation of these plasmids encoded peptides that interfered with activation of PhoB by VanS(Met-Ser). A minimal size fragment (Met-Ile) was shown to be both necessary and sufficient for potent inhibition (85%) of this activation.


INTRODUCTION

Vancomycin is a potent glycopeptide antibiotic that has been used in the treatment of Gram-positive bacterial infections for over 30 years(1) . Vancomycin causes cell death by disrupting cell wall biosynthesis; the drug binds to the terminal D-Ala-D-Ala moieties of the growing peptidoglycan cell wall, thereby inhibiting the transpeptidation and transglycosylation steps necessary for peptidoglycan cell wall maturation(2) . This unique mode of action has made the drug relatively immune to resistance problems; however, over the past decade, an increasing number of highly resistant strains of Enterococcus have been identified in clinical isolates and waste water systems(3) . Vancomycin resistance in these strains requires five plasmid-borne genes: vanH, vanA, vanX, vanR, and vanS(4) . Three of these gene products (VanH, VanA, and VanX) are enzymes that together confer resistance by leading to the incorporation of modified cell wall precursors in the peptidoglycan. VanH is a D-specific alpha-ketoacid reductase that converts pyruvate to D-lactate(5, 6) . VanA is a D-Ala-D-Ala ligase with altered specificity; it is capable of utilizing the D-lactate produced by VanH for efficient D-Ala-D-lactate depsipeptide synthesis(7) . The D-Ala-D-lactate depsipeptide, when incorporated into the peptidoglycan, confers resistance to vancomycin because the drug has a 1000-fold reduced affinity toward D-Ala-D-lactate than toward the native D-Ala-D-Ala peptidoglycan precursors(8) . Recently, VanX has been shown to be a D-, D-specific dipeptidase, which is competent in D-Ala-D-Ala hydrolysis but does not hydrolyze the D-Ala-D-lactate depsipeptide(9, 10) , effectively reducing the pools of the D-Ala-D-Ala dipeptide and shunting flux to D-Ala-D-lactate peptidoglycan termini. All together, these enzymes provide an efficient alternate cell wall biosynthetic pathway that results in the selective incorporation of D-Ala-D-lactate into the growing peptidoglycan cell wall.

VanS and VanR comprise a two-component regulatory system for transcriptional activation of the genes vanH, vanA, and vanX(11, 12) . Two-component regulatory systems are signal transduction pathways commonly used by prokaryotes to sense and adapt to stimuli in the environment; as many as 50 different systems may exist in a simple bacterium such as Escherichia coli, as well as other bacteria(13, 14) . In addition, analogous signal transduction pathways have recently been identified in eukaryotic organisms including yeast(15, 16) , plants (17) , and neurospora(18) . These systems are characterized by a sensor kinase (often a transmembrane signaling kinase such as VanS), which undergoes autophosphorylation on a histidine residue, and this phosphoryl group is then transferred to an aspartate residue on a response regulator protein (in this case, VanR), which usually acts as a transcriptional activator. Like many signaling kinases, VanS has an N-terminal domain with two transmembrane-spanning segments believed to act as a signal sensing domain and a C-terminal cytoplasmic ``transmitter'' domain with autophosphorylation and phosphotransfer activities(13) . Biochemical studies on the cytoplasmic domain of VanS(Met-Ser) have shown that it is readily autophosphorylated at a histidine residue (His) in the presence of ATP and that phospho-VanS is capable of efficient phosphotransfer to an aspartate residue on VanR (Asp) (12) . Genetic studies have implicated VanR as a transcriptional activator of the vancomycin resistance genes(11) . These predictions have been borne out by in vitro studies with the purified protein. Gel mobility shift and DNase footprinting analyses have shown that phospho-VanR has an enhanced binding affinity for the P and P promoter regions of the vancomycin resistance operon(19) .

Sensor kinases and response regulators of two-component regulatory systems share extensive sequence similarities to other family members, even of phylogenetically distant species(13) . These sequence similarities probably lead to structural similarities that are responsible for cross-reactivities between sensor kinases and response regulators of different systems. This phenomenon, called ``cross-talk,'' has been observed in vitro(20, 21) , and it has been implicated in complex phenotypes in vivo. For example, the E. coli phosphate (PHO) regulon is activated by two different sensor kinases in vivo, which may represent a special form of cross-talk called cross-regulation(22) . The sensor kinase PhoR activates the response regulator PhoB that in turn activates synthesis of bacterial alkaline phosphatase in response to limiting phosphate levels (Fig. S1A)(23) . In the absence of PhoR, the sensor kinase CreC or acetyl phosphate activates PhoB ( (24) and (25) and for reviews, see (22) and (26) ) Furthermore, cross-talk interactions have been used to identify new two-component regulatory genes by complementation of known sensor kinase mutants (E. coli envZ and/or phoR creC(27, 28, 29, 30) ). These results reinforce the concept that individual two-component systems share a common mechanism and suggest that it is likely that similar regions of the sensor kinases and response regulators are involved in the transmembrane signal sensing, autophosphorylation, and interprotein phosphorelay steps of these signal transduction pathways.


Figure S1: Scheme 1.



We are working to define the regions of VanS involved in the signaling process, including those involved in the recognition and phosphorylation of its cognate response regulator VanR. Studies on the chemotaxis sensor CheA have demonstrated that there are distinct regions that are responsible for its autokinase and phosphotransfer activities(31) , as well as regions that specifically interact with the response regulator, CheY(32) . To perform a similar molecular dissection of VanS, we developed a genetic method to screen VanS fragments that retain structure/function elements necessary for signal transduction. Since the potential health risks associated with vancomycin-resistant Enterococcus mitigated against using the native VanS/VanR signaling pathway as an assay for VanS function, we constructed an artificial signal transduction pathway that involved in vivo activation of the E. coli response regulator PhoB by VanS through cross-talk. In this system, all of the known signaling pathways for activation of PhoB (PhoR, CreC, or acetyl phosphate synthesis) have been eliminated by mutation(25) , and therefore any activation by VanS can be quantified by measuring bacterial alkaline phosphatase (encoded by phoA) (Fig. S1B). In this report, we demonstrate that the cytoplasmic domain of VanS (residues Met to Ser) is capable of high level activation of E. coli PhoB both in vitro and in vivo, and we have used this cross-reactivity as a reporter system to identify a domain of VanS that inhibits PhoB activation by VanS.


MATERIALS AND METHODS

Plasmid pAT89 carrying vanS was a generous gift from Dr. M. Arthur (Institut Pasteur). pBB100 was a generous gift from Barry Ballard (Harvard Medical School), and pBAD32 was a generous gift of Luz-Marie Guzman and Jon Beckwith (Harvard Medical School). Nucleotide sequencing was performed with the Sequenase kit (U. S. Biochemical Corp.). Oligonucleotides were prepared by Dr. Charles Dahl (Harvard Medical School). Antibiotics were from Sigma and (unless otherwise noted) were used at the following concentrations: ampicillin, 100 µg/ml; chloramphenicol, 34 µg/ml; and kanamycin, 50 µg/ml.

Plasmid Construction

To construct a plasmid suitable for overproduction of the cytoplasmic domain of VanS (Met-Ser), unique NdeI (upstream) and BamHI (downstream) restriction enzyme sites were incorporated using the polymerase chain reaction with the following primers: upstream (Met start codon underlined), 5`-TTGGTACCCATATGACATTAAAACGGACTCTGGAA-3`; downstream, 5`-CAACTATTTTCCTCCAGGATTCCTAGGGAGCTCCC-3` using plasmid pAT89 as a template for vanS(11) . The amplification product was cloned into NdeI- and BamHI-cleaved pET22b (Novagen Inc.) to afford pET22b:vanS(Met-Ser), designated as pSLF1, and verified by DNA sequencing of the vanS segment.

Two kinds of plasmids were constructed to provide controlled synthesis of VanS (Met-Ser). One was constructed using pBB100, a derivative of pSPORT1 (Life Technologies, Inc.) containing a synthetic merR gene(33) . pSPORT1 is a pMB1-based vector carrying P and encodes ampicillin resistance and LacI. The other was constructed using the compatible p15A-based vector, pBAD32. pBAD32 is a derivative of pBAD28 (34) that carries P and encodes both chloramphenicol resistance and AraC. The pSPORT and pBAD plasmids used in this study are described in Table 1. Construction of plasmid pSLF4 was accomplished using polymerase chain reaction with primer (upstream) 5`-TACTTGGCGCAGGATATCAAAACGCCCCTTACATC-3` and the downstream primer from the pSLF1 plasmid construction. The amplification product of these primers was digested with HhaI and BamHI and ligated with two purified fragments of pSLF3 obtained by digestion with NdeI, HhaI, and BamHI. Plasmids pSLF1, pSLF4, and pSLF6 were confirmed by nucleotide sequencing of the vanS segment; all others were confirmed by mapping with restriction enzymes.



Bacterial Strains

All in vivo studies were carried out using E. coli K-12 strain BW20992 or BW21469. Both of these are DE3(lac)X74 phoR68 Delta(creBCD)153 Delta(pta ackA) phn(EcoB). BW21469 is in addition Delta(araCBAD)714 recA1. Both are otherwise isogenic with strain BW13711(35) . Each was made in a series of phage P1 crosses. P1 lysates were prepared on the following strains: BW5206 (srlC300::Tn10), BW7261 (leu-63::Tn10), BW9301 (recA1), BW12070 (Delta(thr creABCD)::Tn5-132), BW12354 (phoA532), BW13635 (proC::Tn5-132), BW14087 (phoB23), BW20891 (Delta(creBCD)153), FD500 (nuoG::Tn10 purF), JP329 (Delta(araCBAD)714), and TA3516 (Delta(ackA pta)). BW5206 and BW9301 are described elsewhere(35) . The Delta(creBCD)153 mutation of BW20891 is deleted from a KpnI fragment internal to the creABCD operon; it was made in vitro and was recombined on the chromosome by allele replacement using an R6Kori vector derived from pWM7(36) . (^1)FD500 was from F. Dailey (Harvard University), and JP329 was from J. Pogliano (Harvard Medical School). TA3516 was described earlier(25) . Other strains are from B. L. Wanner's collection. Each of the relevant mutations was introduced in a two-step procedure in order to avoid the presence of additional undesirable mutations. An ancestral strain was made tetracycline-resistant and sorbitol-, leucine-, proline-, purine-, or threonine-negative using an appropriate P1 lysate. These were then made sorbitol-positive or prototrophic with P1 lysates of BW9301, BW12354, BW14087, BW20891, JP329, or TA3516 in order to introduce the respective linked mutation and to eliminate the tetracycline resistance determinant. P1 transductions were carried out as described elsewhere (35) .

Protein Purification

VanR and MBP^2-VanS were purified as described previously to >95% purity as assessed by Coomassie Brilliant Blue-stained SDS-PAGE gels(12) . Purified PhoB (>95%) was a generous gift from Soo-Ki Kim.^1

Preparation of Phospho-MBP-VanS and Phosphotransfer Studies

MBP-VanS (4 nmol) was incubated with [-P]ATP (1 mM, 100 mCi) in 100 µl of autophosphorylation buffer (50 mM Tris, pH 7.4, 50 mM KCl, and 1 mM MgCl(2)). After 1 h, the reaction mixture was diluted with 500 µl of buffer A (50 mM Tris, pH 8.0, 50 mM NaCl, 5 mM EDTA) and then concentrated to approx20 µl using a Microcon-10 concentrator (Amicon Co.). This process was repeated 5 times to yield P-labeled MBP-VanS (2 nmol, 7500 cpm/nmol) free from excess [-P]ATP. Phosphotransfer from P-labeled MBP-VanS to PhoB or VanR was initiated by the addition of P-labeled MBP-VanS (0.5 nmol) to a solution of either PhoB (2 nmol) or VanR (1 nmol) in 50 µl of autophosphorylation buffer. At designated time points, aliquots (6 µl) of each mixture were quenched with SDS-stop buffer (6 µl of 125 mM Tris, pH 6.8, 2.5% SDS, 2 mM EDTA, 0.0025% bromphenol blue, and 25% glycerol). The phosphorylated proteins were separated using 12.5% SDS-PAGE and quantified using phosphorimage analysis (Molecular Dynamics Inc.).

Bacterial Alkaline Phosphatase Activity Measurements

Cells were grown on agar of the same media composition but lacking an inducer and then inoculated into broth cultures with or without IPTG or arabinose and grown at 37 °C for 16 h. Triplicate cultures were always grown from separate colonies. Bacterial alkaline phosphatase activities were measured as described previously(35) . Units are nmol of p-nitrophenol produced per min at 37 °C normalized to cell culture density measured at 420 nm.

Transposon Mutagenesis

Plasmid pSLF3 was subjected to m transposon mutagenesis using the liquid culture protocol of Berg et al.(37) . Colonies with mutant plasmids were selected on LB agar containing ampicillin, kanamycin, and nalidixic acid (20 µg/ml). Approximately 10,000 colonies were pooled, and the plasmid DNA was isolated.

Screen for Cross-talk Inhibition by Plasmid Mutants of the pSLF3::m Library

Aliquots of the pSLF3::m library DNA were transformed into strain BW21469 carrying pSLF5 and grown on TYE agar (1% tryptone, 0.5% yeast extract, 0.8% NaCl) containing ampicillin and chloramphenicol. To survey effects due to VanS synthesis, all media contained 0, 50, or 100 µM arabinose and 0 or 1 mM IPTG in addition to 5-bromo-4-chloro-3-indolyl-phosphate as an indicator for bacterial alkaline phosphatase activity. Transformants that were nearly white in the presence of arabinose and IPTG were chosen for further study. These were retested on each of the other media. Plasmid DNAs were isolated from suspected mutants and transformed into DH5alpha. Plasmid DNA was isolated from those colonies conferring only ampicillin resistance, and the transposon insertion sites were determined by restriction enzyme mapping or nucleotide sequencing using the m res and kan primers(37) . Also, alkaline phosphatase phenotypes were confirmed after re-transformation into BW21469 carrying pSLF5. These transformants were used for broth culture enzyme assays.


RESULTS

Activation of the Phosphate Regulon by Expression of VanS

In order to test activation of PhoB (the PHO regulon response regulator and transcriptional activator of the alkaline phosphatase structural gene phoA) by VanS in vivo, a plasmid (pSLF3) was constructed in which synthesis of the VanS cytoplasmic domain (Met-Ser) is controlled by the regulated promoter P. Activation of PhoB in vivo and subsequent phoA expression (Fig. S1) may result from phosphorylation by the sensor kinases PhoR or CreC or in response to acetyl phosphate synthesis in E. coli; however, none of these mechanisms occur in BW20992 due to their loss by mutation(25) . Transformation of pSLF3 into BW20992 resulted in elevated levels of alkaline phosphatase synthesis even in the absence of IPTG; a 10-fold activation is seen without inducer (Table 2). This effect was not observed in transformants carrying the vector pBB100, indicating that the increased alkaline phosphatase synthesis is due to the presence of VanS (Met-Ser). The elevated activity under these conditions is attributable to a moderate basal level of VanS(Met-Ser) synthesis from pSLF3. Much higher levels of alkaline phosphatase synthesis were observed under conditions of induction. A approx350-fold increase in alkaline phosphatase activity was measured in the presence of IPTG.



Alkaline phosphatase synthesis in BW20992 is due to activation of PhoB by VanS. No activation was observed when a phoB or phoA mutant was transformed with pSLF3, even under conditions of induction (Table 2). Furthermore, activation of PhoB by VanS was abolished by PhoR in the presence of P(i) (Table 2). This was expected because PhoR inhibits activation of PhoB when P(i) is in excess, presumably by facilitating the dephosphorylation of phospho-PhoB. These results suggest that in strain BW21096, the net dephosphorylation by PhoR is more efficient than the phosphotransfer to PhoB from VanS (Fig. S2).


Figure S2: Scheme 2.



A further test of the cross-talk between VanS and PhoB in vivo was performed using a site-directed mutation of the presumed site of autophosphorylation in VanS (His). Transformation of BW20992 with a plasmid (pSLF4) encoding the H164Q form of VanS(Met-Ser), resulted in no increased activation of alkaline phosphatase synthesis (Table 2). This is to be expected if activation of PhoB is due to phosphoryl transfer from phospho-VanS. Taken together, these results are consistent with an efficient level of cross-talk between VanS and PhoB in vivo, in which VanS activates the PHO regulon through direct phosphorylation of the PHO response regulator PhoB. This level of PhoB activation is similar to that observed with its partner sensor kinase PhoR, as comparable strains synthesize about 600 units of alkaline phosphatase upon activation of PhoB by PhoR under conditions of P(i) limitation. (^3)

Phosphotransfer from MBP-VanS to PhoB

In order to characterize the phosphotransfer efficiency of VanS to PhoB, phosphorylation experiments were carried out in vitro using purified proteins. These studies utilized an MBP-fusion to the VanS cytoplasmic domain since this protein can be readily purified and has been characterized previously with its cognate response regulator, VanR (12) . As expected, both VanR and PhoB show no evidence of phosphorylation with ATP over time (Fig. 1, lanes1 and 2). However, rapid phosphotransfer is observed upon incubation of PhoB with autophosphorylated VanS; most of the VanS phosphate is transferred to PhoB within 5 min (lane5). With VanR, quantitative transfer is observed (lane4) on this time scale(12) . As expected, the phosphotransfer from VanS to PhoB was inhibited by EDTA (data not shown). A direct comparison of the time course of phosphoryl transfer from phospho-VanS to PhoB (Fig. 2A) or VanR (Fig. 2B) was performed by using purified P-labeled MBP-VanS free from ATP--P. A t of 10 min was seen for PhoB with a 4:1 PhoB/VanS stoichiometry versus an estimated 10 s for VanR with a 2:1 VanR/VanS stoichiometry. By this measure, phosphoryl transfer from phospho-VanS to PhoB is at least 100-fold less efficient than from phospho-VanS to its cognate VanR.


Figure 1: Phosphorylation of response regulators by phospho-MBP-VanS. Lanes1-3, proteins were incubated in 15 µl of phosphorylation buffer (50 mM Tris, 50 mM KCl, 1 mM MgCl(2), pH 7.4) for 60 min at 37 °C with [-P]ATP (final concentration, 1 mM; 50 µCi/pmol) and then quenched with 10 µl of SDS stop solution. Final protein concentrations were as follows: VanR, 20 µM; PhoB, 20 µM; VanS, 5 µM. Lanes4 and 5, MBP-VanS (32 µM) was incubated with [-P]ATP (final concentration, 1 mM; 50 µCi/pmol) for 60 min in phosphorylation buffer at 37 °C, and then 2 µl of this mixture were added to 12.5 µl of VanR (lane4, 24 µM) or PhoB (lane5, 24 µM) solutions in phosphorylation buffer. After 5 min at 30 °C, the reactions were quenched with 10 µl of SDS stop buffer. Samples of the quenched reactions (12.5 µl) were immediately loaded on a 12.5% SDS-PAGE gel. The phosphorimage (Molecular Dynamics Inc.) of the gel is shown.




Figure 2: Time Course of Phosphotransfer from P-MBP-VanS to (A) PhoB and (B) VanR. Phosphorimages of SDS-PAGE gels of the phosphorylated proteins are shown, conditions are described under ``Materials and Methods.'' Protein concentrations were as follows: MBP-VanS, 10 µM; VanR, 20 µM; PhoB, 40 µM.



Titration of PhoB Activation Using pBAD:VanS with Arabinose

The above results show that VanS(Met-Ser) leads to efficient activation of genes belonging to the PHO regulon in vivo and that MBP-VanS leads to efficient phosphorylation of the PHO regulon transcription factor PhoB in vitro. To define domains of VanS that may be important for these interactions, additional plasmids showing regulatable synthesis of VanS were constructed. In these plasmids, VanS(Met-Ser) is synthesized under the control of the arabinose-regulatable promoter P. These plasmids are also compatible with those that synthesize VanS from P. Accordingly, doubly transformed derivatives of a reporter strain carrying one form of vanS under P control and a different form of vanS under P control are expected to synthesize (primarily) one protein or both, depending upon the presence of IPTG and arabinose. In addition, a new reporter strain (BW21469) was constructed to accommodate these new plasmids. This strain is deleted of the araCBAD operon, thus allowing for tighter regulation of P. Also, this strain is recA, so two different vanS plasmids may be maintained in the same strain. It was felt that the construction of these materials may provide a powerful genetic reporter system for the identification and characterization of regions of VanS important for its interaction with PhoB.

As expected, synthesis of the VanS(Met-Ser) under arabinose control in plasmid pSLF5 resulted in high levels of alkaline phosphatase synthesis (Table 2). Arabinose caused a approx500-fold increase in alkaline phosphatase activity. No detectable synthesis was observed in the absence of arabinose, thus showing the tighter control of the P promoter in comparison with P. This tight control is further substantiated in Fig. 3, where the amount of alkaline phosphatase activity is shown to vary with the concentration of arabinose. A similar titration curve has been observed for the expression of other genes in pBAD vectors(34) . A linear increase in alkaline phosphatase activity is observed with increasing concentrations up to about 100 µM arabinose. All experiments involving pBAD plasmids were carried out using fructose as a carbon source in order to overcome problems resulting from catabolite repression (data not shown).


Figure 3: Titration of PhoB Activation with pSLF5. Transformants of strain BW21469 carrying pSLF5 were grown on 2XYT agar containing chloramphenicol (100 µg/ml) and then inoculated in 5 ml of 2XYT broth containing chloramphenicol and grown to saturation overnight at 37 °C with aeration. A sample of the cell culture (200 µl) was diluted in 20 ml of TYE medium containing chloramphenicol, and 0.5-ml portions were subcultured in broth containing arabinose. Triplicate cultures were assayed for each concentration. Cell cultures were grown for 8 h at 37 °C with aeration and then assayed for alkaline phosphatase activity as described under ``Materials and Methods.''



Preparation of C-terminal Truncation Fragment Library Using Transposon Mutagenesis

On the premise that a VanS fragment capable of interacting with PhoB may inhibit the activation of PhoB by VanS(Met-Ser), we constructed a mutant library of the vanS plasmid pSLF3 using the m transposon(37) , a Tn1000 derivative. This transposon was chosen because it is relatively small and it leads to a near random distribution of insertion sites. Also, analysis of the m sequence reveals translational stop codons in all three reading frames for insertions of m in both orientations. Therefore, all insertions into the vanS coding region are expected to lead to formation of C-terminal truncations of the open reading frame. The transposon sequence introduces fusions to the C-terminal truncations (Table 3); however, only two kinds of insertions result in fusions >25 residues. After transposon mutagenesis of pSLF3, approx10,000 colonies were isolated. To test for the randomness of insertion sites among these, 20 colonies were isolated, and the insertion sites were mapped by digestions with BamHI and KpnI. All of these appeared to map to unique sites; 20% were located within the vanS coding region. This frequency is expected since vanS corresponds to 20% of the nonessential regions of the plasmid; insertions into bla were counter-selected by selecting ampicillin-resistant colonies. We screened this mutant library for plasmids synthesizing VanS peptides capable of activating PhoB by a transphosphorylation mechanism or capable of interfering with PhoB activation by VanS.



Screen of VanS C-terminal Truncation Plasmid Library for Fragment-based Activation of PhoB

Our first screen involved testing for whether a subpopulation of VanS fragments would contain the necessary elements for phosphorylation and subsequent phospho-relay to PhoB. Therefore, we screened the mutant library for fragments capable of complementing the VanS(Met-Ser) H164Q mutant. This mutant is unable to undergo autophosphorylation, but it would be expected to phosphorylate VanS fragments containing the phosphorylation site (His) in trans(38) , and therefore any activation of the PhoB would require a phospho-VanS fragment intermediate. In this screen, the mutant plasmid library was transformed into BW21469 carrying pSLF6, and transformants synthesizing VanS fragments capable of PhoB activation would be recognizable as blue colonies on bacterial alkaline phosphatase indicator agar (containing 5-bromo-4-chloro-3-indolyl-phosphate) due to increased synthesis of alkaline phosphatase. In spite of screening more than 3000 transformants under conditions where the expression of VanS(Met-Ser) and the m transposon library were varied, we were unable to identify plasmids encoding a fragment able to activate PhoB in this exhaustive search.

Screen of vanS C-terminal Truncation Plasmid Library for Inhibition of VanS(Met-Ser)/PhoB Signaling

Our second screen involved testing for VanS fragments that may interfere with activation of PhoB. In this case, we used a reporter system based on the activation of PhoB by VanS(Met-Ser) to screen the vanS C-terminal truncation library for those encoding a fragment that interferes with this activation. To allow for the prospect that inhibition may result only if the VanS truncation fragment were synthesized in greater amounts, the screen was carried out under conditions of partial induction of VanS(Met-Ser) synthesis by the arabinose-regulated plasmid pSLF5. The mutant library was transformed into a BW21469 derivative carrying pSLF5, and doubly transformed colonies were selected on bacterial alkaline phosphatase indicator agar containing ampicillin, chloramphenicol, kanamycin, 5-bromo-4-chloro-3-indolyl-phosphate, 1 mM IPTG, and 20, 50, or 100 µM arabinose. Under these conditions, about 35% of the colonies appeared to be less blue. About 50% of the colonies were white when the same library was transformed into BW21469 without pSLF5, indicating a high proportion of pSLF3::m library had insertions within vanS. A sample of 18 pSLF3::m plasmids that appeared to interfere with activation of PhoB by VanS in this assay were examined. These plasmids were characterized by restriction mapping and nucleotide sequencing to determine the transposon insertion site. All of them had a single, unique insertion, but two of these encoded an identical fragment (Met-Ile). The distribution of the insertion sites over the vanS coding region was nongaussian with a distinct clustering near the first third of the gene (Fig. 4). These data suggest that a minimal fragment length of approx80 residues is necessary and sufficient to interfere with activation of PhoB by VanS. As expected, longer fragments containing up to 240 residues were also able to inhibit activation. All of the fragments that interfere with the PhoB activation by VanS contain His, the site of autophosphorylation in VanS.


Figure 4: Schematic of inhibitory fragments of VanS identified using the C-terminal truncation library. The VanS cytoplasmic domain (Met-Ser) is shown schematically with the conserved regions as described by Parkinson and Kofoid (13) annotated. Regions containing VanS sequence are denoted as darkbars; lightregions represent sequences added as in-frame fusions from the transposon insertions.



Plasmids encoding four of these fragments were assayed for effects on alkaline phosphatase synthesis in broth cultures in order to quantify the amount of inhibition (Fig. 5). These plasmids were chosen as representatives of the full range of VanS fragment sizes identified from the genetic screen, ranging from 80 to 241 amino acids of VanS. All of these plasmids demonstrated inhibition both in the presence or absence of IPTG for induction of the fragment synthesis (Fig. 5). The inhibition observed in the absence of IPTG is likely to be due to the moderate basal level of expression from the pSPORT plasmid, which was also observed with the full-length VanS construct (pSLF3, Table 2). In all cases, the inhibition was greater with increased level of fragment synthesis, and the extent of inhibition ranged from approx50% to 85%. No correlation to the relative amount of inhibition and the fragment size was observed.


Figure 5: Inhibition of alkaline phosphatase synthesis by C-terminal truncated fragments of VanS. Alkaline phosphatase assays were performed as described under ``Materials and Methods.'' Fragments denoted with an asterisk contain an 11-amino acid fusion from the transposon, those with contain a 27-amino acid fusion (see Table 3). Assays performed without induction (0 mM IPTG) of the fragment synthesis are openbars, data for induced (1 mM IPTG) fragments synthesis are represented by filledbars.




DISCUSSION

The two-component VanS and VanR proteins of vancomycin resistance plasmids in Gram-positive Enterococci control transcription of the structural genes vanH, vanA, and vanX that encode enzymes that produce a modified peptidoglycan structure with 1000-fold lower affinity for vancomycin. Efforts to study the interactions of VanS and VanR in E. coli await a good method for transcriptional activation of the vanH and vanR promoters in Gram-negative bacteria. The studies reported here establish that the cytoplasmic domain (Met-Ser) of the VanS sensory autokinase can recognize the response regulatory component PhoB of the E. coli PHO regulon and drive transcriptional activation of phoA (encoding alkaline phosphatase). This heterologous recognition between sensor and response regulator opens the possibility of structure/function mutagenesis in both VanS and PhoB to screen for mutual recognition domains and should also provide an assay for evaluation of the full-length transmembrane VanS function, especially in regard to the ligand(s) that promotes VanS activation, e.g. in E. coli mutants with a leaky outer membrane envelope(39) .

The in vivo cross-talk between VanS and PhoB was validated by in vitro analysis of phosphoryl group transfer from the phosphohistidine form of MBP-VanS to PhoB with the purified proteins. Comparison of the rates of phosphoryl transfer from phospho-MBP-VanS to purified PhoB or VanR when the response regulator proteins are in excess of VanS (1:4 molar ratio for PhoB, 1:2 molar ratio for VanR) indicates a 100:1 rate preference for homologous (VanR) versus heterologous (PhoB) phosphorylation of the response regulator. K(m) and K(D) values are yet to be obtained to provide estimates of k/K(m) catalytic efficiency criteria. Both the in vivo and in vitro VanS/PhoB assays may be applicable to screen for agents that would inhibit two-component regulatory system signal transduction (40) and ultimately inhibit vancomycin activation of the complex vanRSHAX operon.

The observed cross-reactivity between VanS and the E. coli PHO regulon provides an excellent in vivo assay for VanS function. This system may be exploited to investigate the underlying mechanisms of VanS signaling, and it may also provide a means for a detailed understanding of the molecular interactions that govern the signaling processes in two-component systems in general. Towards this goal, we performed two screens of a genetic library of VanS fragments to identify those regions of VanS involved in the transmembrane signaling and phospho-relay steps of the signal transduction pathway. First, a search was conducted for VanS fragments that retain the phosphotransfer activity to PhoB, and, second, a search was conducted for VanS fragments that inhibit the VanS/PhoB phosphoryl transfer. To this end, we prepared and screened mutant libraries of the cytoplasmic VanS construct that were further truncated from the C terminus by m transposon mutagenesis. In the first search, we assayed the library for fragments capable of complementing a VanS(Met-Ser) mutant (H164Q) that would be expected to phosphorylate fragments in trans but is unable to undergo autophosphorylation. No subfragments of the VanS(Met-Ser) construct were detected that retained the ability to activate PhoB. This negative screening result may not be surprising since fragment-based PhoB activation would require efficient phosphorylation of the fragment in trans from the VanS(Met-Ser) H164Q mutant as well as efficient transfer of the phosphoryl group to PhoB.

To assess the ability of VanS fragments to inhibit VanS(Met-Ser) interaction with PhoB required simultaneous synthesis of both VanS(Met-Ser) and VanS subfragments, resulting from the m transposon insertion library. Several mutant plasmids were identified and sequenced that yielded a nested set of fragments with progressive truncations from the C terminus. The minimal size VanS fragment that retains inhibition contains 80 residues (Met-Ile). There is precedent in the E. coli chemotaxis two-component regulatory system for dominant-negative phenotypes resulting from the overexpression of the sensory kinase CheA (32) and the serine chemoreceptor Tsr (41) in a ``domain liberation'' approach. In the VanS case, this small 80-amino acid fragment retains His that is the autophosphorylation site and the phosphoryl donor (to VanR and PhoB). While the small size (9 kDa) augurs well for structural determination of the dominant negative VanS(Met-Ile) fragment, initial attempts to overproduce it have led to insoluble protein that has so far resisted functional renaturation. It will be important to determine whether the 80-amino acid inhibitory fragment is interfering with VanS autophosphorylation from ATP, e.g. perhaps by heterodimerization with VanS (Met-Ser), or by reducing the levels phospho-PhoB, or both. In order to test whether the fragments inhibit the VanS/PhoB signaling by affecting the levels of phospho-PhoB in the cell, e.g. by blocking the interaction with VanS(Met-Ser) or by aiding in the dephosphorylation of phospho-PhoB, we expressed the fragment under conditions of constitutive PhoB activation by CreC. No effect was observed on the PhoB activation under these conditions (data not shown). These negative results imply that the fragments inhibit the signal transduction pathway by interfering with the autophosphorylation of VanS (Met-Ser). It remains to be determined if further truncation from the N terminus of VanS(Met-Ser) will lead to a dominant negative phenotype. In this regard, the osmosensor kinase EnvZ (42) is thought to have a similar overall topology to VanS, and a ``linker region'' between the transmembrane region and the histidine phosphorylation site of EnvZ has an important role in ``locking in'' fixed signaling modes. Similar linker regions have been noted in the related chemoreceptor proteins (14, 43) and the genetic hybrid protein Taz-1(44) . This linker region is also retained in the Met-Ile fragment of VanS.

In summary, these approaches may advance our understanding of homologous and heterologous sensor/response regulator recognition elements for binding and phosphotransfer; for antibiotic and antifungal agents that may disrupt these systems in bacteria, yeast, and fungi; and in particular the specific reversal of vancomycin resistance in pathogenic Gram-positive bacteria.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM35392 (to B. L. W.) and GM49338 (to C. T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Institutes of Health Postdoctoral Fellowship (NIH GM16259).

To whom correspondence should be addressed.

(^1)
S.-K. Kim and B. L. Wanner, unpublished results.

(^2)
The abbreviations used are: MBP, maltose binding protein; PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl-1-thio-beta-Dgalactopyranoside; MOPS, 3-(N-morpholino)propanesulfonic acid.

(^3)
W. Jiang and B. L. Wanner, unpublished results.


ACKNOWLEDGEMENTS

We thank Eugenio I. Vivas (Harvard Medical School) for assistance with the transposon mutagenesis, Barry Ballard for helpful discussions and the plasmid pBB100, and Soo-Ki Kim (Purdue University) for purified PhoB protein.


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