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Originally published In Press as doi:10.1074/jbc.M504464200 on October 27, 2005

J. Biol. Chem., Vol. 280, Issue 51, 42423-42432, December 23, 2005
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Analysis of RovA, a Transcriptional Regulator of Yersinia pseudotuberculosis Virulence That Acts through Antirepression and Direct Transcriptional Activation*{boxs}

Hien J. Tran{ddagger}, Ann Kathrin Heroven{ddagger}, Lars Winkler§, Thomas Spreter§, Birgitta Beatrix§, and Petra Dersch{ddagger}1

From the {ddagger}Junior Research Group 6, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany and the §Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany

Received for publication, April 25, 2005 , and in revised form, October 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Enteropathogenic Yersinia species are Gram-negative bacteria that can cause food-borne infections in animals and humans (yersiniosis), with symptoms such as self-limiting enteritis and mesenteric lymphadenitis (1, 2). During infection Yersinia penetrates and crosses the epithelial layer of the intestine through M-cells and colonizes underlying lymphatic tissues (3). The outer membrane protein invasin is the most efficient factor that promotes uptake of the bacteria into M-cells (4, 5).

Expression of the chromosomally encoded invasin gene is controlled by the transcriptional activator RovA (6, 7). Maximal RovA-mediated transcription of the invasin gene inv requires the binding of RovA to two AT-rich sequences located in a DNA segment extending 200 bp upstream of the transcriptional start site of the inv gene (8). RovA also functions as an activator of its own transcription. It interacts directly and specifically with sequences far upstream in the rovA regulatory region, and this interaction is required for full activation of two different rovA promoters located 76 nt2 (P1) and 343 nt (P2) upstream of the translational start site (8). Transcription of the rovA gene and the RovA-dependent inv gene are both subject to silencing by the nucleoid-associated H-NS protein. The binding sites of H-NS and RovA are superimposed within the inv and rovA promoters, indicating that RovA might act as an antirepressor (8). Recently, it has become evident that RovA is involved in the regulation of multiple genes of Yersinia in addition to inv and rovA, some of which seem to play an important role in pathogenesis (6, 9).

RovA analogous proteins (SlyA/Hor/Rap) are also present in other pathogenic Enterobacteriaceae. They regulate a wide range of physiological processes involved in survival, stress adaptation, and virulence. In Salmonella, SlyA regulates virulence factors necessary for environmental adaptation and survival in mice (10-13). SlyA of Escherichia coli induces a cryptic clyA gene, encoding a contact-dependent pore-forming cytolysin of the RTX toxin family. RovA homologs, Rap and Hor, in Serratia marcescens and the plant pathogen Erwinia carotovora regulate the production of secondary metabolites, such as antibiotics, pigments, and exoenzyme virulence determinants (14-16). The RovA/SlyA proteins are very closely related in sequence and are, in part, heterogenically cross-functional (16).

Comparison of amino acid sequences in genomes revealed that the RovA/SlyA proteins share low level sequence similarity (≤40% identity) with a large number of different transcriptional regulators identified in diverse bacterial and archaeal genomes (supplemental Fig. A). A prototypic member of the entire regulator family is MarR, a repressor of the marROAB operon mediating intrinsic multiple antibiotic resistance in E. coli (17, 18). To date, the crystal structures of three MarR-like proteins have been determined without their DNA targets. Despite their low amino acid sequence similarity, all three family members show a similar overall structure. They form dimers and share a common internal fold with a conserved winged-helix motif that is believed to mediate interactions with DNA (19-21). Despite this, MarR-like proteins seem to retain a very flexible and highly adaptable protein fold and show significant divergence at their terminal ends, which apparently allow them to fulfil different functions (21).

Phylogenetic analysis indicates that the RovA/SlyA proteins, which have the potential to activate gene transcription, represent a subgroup placed in a separate phylogenetic tree between other MarR-like subfamilies, which typically repress gene transcription. In this context it is important to note that, unfortunately, several members of other MarR-type subgroups with distinct functions and very low homologies (<25%) to the RovA/SlyA family were also named SlyA, e.g. the crystallized protein SlyAEF of Enterococcus faecalis and SlyA2 (Hos) of E. coli (supplemental Fig. A) (21-23).

A three-dimensional structure of a RovA/SlyA family member is not yet available, and specific details about the structural organization and the oligomeric state remain unclear. In addition, little is known about the interaction of this RovA/SlyA subfamily with DNA and the transcriptional machinery, in particular RNA polymerase. For this reason, this study was aimed to define the functional domains of the RovA protein and to investigate direct transcriptional activation of the inv and rovA promoters by this transcription factor.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Media, and Growth Conditions—The strains used in this study are listed in supplemental Fig. B. Overnight cultures of E. coli were routinely grown at 37 °C under aeration in LB medium plus appropriate antibiotics.

DNA Manipulations and Sequence Analysis—All DNA manipulations and transformations were performed using standard genetic and molecular techniques (24, 25). T4 ligase and DNase I were obtained from Roche Applied Science; all restriction enzymes were obtained from New England Biolabs. Oligonucleotides were purchased from Metabion. Taq polymerase (New England Biolabs) and Pfu polymerase (Promega) were used for amplification by PCR. PCR products were purified with the QIAquick PCR purification kit (Qiagen). Sequencing reactions were carried out by Agowa (Berlin, Germany). Radiolabeled [{alpha}-32P]UTP (800 Ci mmol-1) was purchased from PerkinElmer Life Sciences. Plasmids used in this study are listed in Supplemental Fig. B. The rovA gene and its mutant alleles were amplified by PCR and cloned into vectors pQE or pET28a for the synthesis of His6-tagged proteins, generating the pHis series. Plasmids pHT102, pHT103, and pHT104 used for in vitro transcription experiments are derivatives of pJL28 carrying PCR generated DNA fragments of Y. pseudotuberculosis harboring different segments of the inv (pHT102: -207 to +101) or the rovA regulatory region (pHT103: -455 to -114; pHT104: -455 to +96).

Random and Site-directed Mutagenesis of the rovA Gene, Construction of N- and C-terminal Deletions—Random chemical mutagenesis with hydroxylamine of pGN12 (rovA+) was performed as previously described (26). Ten plasmid samples were mutagenized independently and transformed into E. coli pAK002, encoding a RovA-dependent invphoA reporter fusion. White/light blue colonies on LB plates with X-P were isolated and analyzed.

N-terminal deletions and site-directed mutagenesis to introduce alternative amino acid in RovA were performed by a four-primer/two-step PCR procedure of pGN12 with two common external and two internal mutagenesis primers. For C-terminal deletion alleles, PCR fragments were amplified using a universal upstream and a mutagenesis-specific downstream primer. The resulting fragments encoding the rovA gene with the desired mutations were cloned into vector pBAD33 cut with KpnI and XbaI.

Gel Electrophoresis and Western Blotting—Cell extracts of identical amounts of E. coli bearing pBAD33-derivatives encoding the RovA wild-type or various mutant proteins were prepared and separated on a 15% SDS-polyacrylamide gel (24, 25). For the immunological detection of the RovA proteins, the samples were transfered onto an Immobilon-P membrane (Millipore) and probed with a polyclonal RovA-specific antiserum as described (8).

Expression and Purification of RovA and H-NS—The RovA wild-type protein, the RovA mutants and the H-NS protein of Y. pseudotuberculosis with a His-tag at the C-terminal end were expressed in E. coli and purified from 1-liter cultures via nickel-nitrilotriacetic acid agarose (Qiagen) as described in previous studies (7, 8). Protein concentration was determined with the Bradford assay (Bio-Rad). The purity of the RovA proteins and H-NS was estimated to be >95%.

DNA Binding Studies—For the DNA retardation assays a DNA fragment encoding a RovA-binding site of the inv regulatory region (-223 to +90) was generated by PCR. The binding of RovA or RovA mutants to the PCR fragment and separation of the DNA-RovA complex on a 4% polyacrylamide gel was carried out as described (7, 8). For DNase I footprinting, an inv promoter fragment (-276 to +224) was amplified by PCR with a digoxigenin-labeled primer (5'-digoxigenin-ACCATCAGGATTAATGCGG-3') and a non-labeled primer (5'-ATTCCTTATCAAGAGAAACTC). PCR fragments were incubated with the purified RovA protein, digested with DNase I, and the resulting products were separated and visualized as described (8). The protected bands were identified by comparison to a DNA sequence ladder generated with the same DIG-labeled primer used for the amplification of the fragment by PCR. For double-stranded sequencing pPD264 was used as template.

Run-off Transcription Assay—Linearized plasmids pHT102, pHT104, and pHT103 were used as templates for single-round in vitro transcription. Binding reactions contained 4 µl of each DNA template (20 nM), 4 µl of increasing concentrations of RovA and/or H-NS (0.3-1.0 µg) in buffer A (10 mM Tris-HCl, pH 8, 0.1 mM EDTA, 10 mM MgCl2, 200 mM KCl, 50% glycerol), or buffer A alone (control) were kept at room temperature for 20 min. 3.5 µl (0.31 unit/µl) RNA polymerase (RNAP) holoenzyme of E. coli (Epicenter) in buffer B (40 mM Hepes, pH 8, 10 mM MgCl2, 100 mM potassium glutamate) was added to 8 µl of this mixture, previously incubated at 37 °C for 5 min. Elongation was started by the addition of 3.5 µl of prewarmed rNTPs (1 mM ATP, CTP, and GTP, 50 µM UTP, 1 µCi of [{alpha}-32P]UTP, and 500 µgml-1 heparin) and allowed to proceed for 6 min. The reactions were stopped with a form-amide solution containing 20 mM EDTA, 0.25% xylene cyanol and bromphenol blue. Samples were heated to 70 °C for 5 min before loading on a 6% denaturing sequencing gel with a radiolabeled size marker fragment. To generate the size marker, a 135-bp DNA fragment (20 pmol) was radiolabeled in a 20-µl reaction mix, containing 2 µl of T4 polynucleotide kinase (Promega) and 2 µl of [{gamma}-32P]ATP (3000 Ci mmol-1). The labeling reaction was incubated for 30 min at 37 °C and stopped by addition of 1 µl of 0.5 M EDTA, pH 8. Radiolabeled transcripts were quantified on a Fuji phosphorimager and normalized against vector-encoded 106 and 108-nt RNA I transcripts present in each lane.

Cross-linking and Gel Filtration of Purified RovA—For cross-linking, 3-12 mM N-({alpha}-maleimidoacetoxy)succinimide ester (AMAS) or form-aldehyde (0.05-0.1%) in the presence of 2 units of RNAP (Epicenter) was added to 200 ng of purified RovA protein in 25 mM HEPES, pH 7.0, 1mM EDTA. Reactions were incubated for 30 min at room temperature and stopped by adding ethanolamine, pH 7.4, in a final concentration of 50 mM. Subsequently, the samples were analyzed by immunoblotting as described (8). To analyze the oligomerization state of the RovA protein, purified RovA protein was subjected to gel filtration chromatography on a Superdex 75 HR 10/30 column (Amersham Biosciences). The column was equilibrated with 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazol, 1 mM MgCl2, and calibrated using a set of different proteins with known molecular sizes: aprotinin, 6.5 kDa; ribonuclease A, 13.7 kDa; chymotrypsinogen A, 25 kDa; ovalbumin, 43 kDa; albumin, 67 kDa. 200-µl samples of the purified RovA protein (1.4 mg ml-1) were applied to this column run by the Akta 100 Explorer system (Amersham Biosciences). The elution profile was monitored by UV absorption and visualized with the Unicorn 3.0 program (Amersham Biosciences).

Alkaline Phosphatase Assays—Alkaline phosphatase activity was measured in permeabilized cells as described (7, 8). The activities were calculated as follows: OD420·6.46·OD578-1·{Delta}t (min)-1·volume (ml)-1.



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  		FIGURE 1.
Oligomerization state of the RovA proteins. A, in vitro cross-linking of the RovA wild-type protein. Purified protein was incubated without (-) or with 3, 6, and 12 mM AMAS. Cross-linked products were added to SDS sample buffer and separated on a 15% SDS-polyacrylamide gel and detected by immunoblotting with polyclonal anti-RovA antiserum. The molecular mass standard is shown on the left. The RovA monomer and the cross-linked dimeric RovA complexes are indicated by arrows at the right. B, size fractionation of purified RovA on a Superdex 75 column. The elution profile of the RovA protein is shown, monitored by its absorbance at 280 nm. Marker proteins are as described under "Experimental Procedures." The peak fractions of the marker proteins and the void sample (Vo: blue dextran 2000) are indicated by arrows.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oligomerization State of the RovA Protein—The homology of RovA to homodimeric members of the MarR family of transcriptional regulator proteins (supplemental Fig. A) (19, 20) and the fact that RovA protects multiple binding sites in the inv and rovA promoter region (8) suggested that RovA might act as an oligomer. Despite this finding, a non-palindromic consensus sequence has been derived from RovA-protected DNA regions (8), making homotypic RovA-RovA interaction questionable. To assess the ability of RovA to form oligomers we performed in vitro cross-linking experiments with the purified RovA protein using AMAS and analyzed the samples by Western blotting using anti-RovA antibodies. Treatment with the cross-linking agent generated species of RovA with the size of a homodimer (35 kDa) (Fig. 1A). To confirm this result, the purified RovA protein was also size fractionated on a Superdex 75 column. No protein was detected of predicted molecular mass <25 kDa, where monomeric RovA with an apparent molecular mass of 17.2 kDa would be expected to fractionate, but a peak elution occurred in a region predicted to contain proteins of about 35 kDa (Fig. 1B). This demonstrated that the majority of the RovA protein in the solution is present as homodimer.

Direct Activation of rovA and inv Transcription by RovA—In our previous study we demonstrated that the RovA regulatory protein is required for the expression of the inv gene. However, RovA was dispensable for inv transcription when the negative modulator protein H-NS was absent, indicating that RovA acts as an antirepressor (8). Since RovA caused an additional increase in inv expression even in the absence of H-NS, it was assumed that RovA may also have the capacity to stimulate RNAP directly (8). To assess the capability of RovA to stimulate transcription of the inv and rovA promoter(s), we performed in vitro transcription assays with RNAP of E. coli (Epicenter), which is almost identical to that of Y. pseudotuberculosis. Purified RovA and linearized plasmids were employed that carry the entire inv promoter fragment (pHT102) or harbor different portions of the rovA regulatory region (pHT103, pHT104) to analyze transcription from P1rovA and P2rovA, respectively. The amount of the inv and rovA transcripts was expressed relative to the level of the RNA I transcripts, which are produced from the rna I gene encoded on the same plasmids. As shown in Fig. 2, inv and rovA specific run-off transcripts of the expected size were detected with RNAP alone. In the case of the inv promoter (Fig. 2A) only a single transcript was found, whereas additional minor transcripts were detected for rovA (Fig. 2, B and C), indicating that RNAP formed productive complexes at closely adjacent nucleotides in vitro. Addition of increasing amounts of the RovA protein in the presence of RNAP further stimulated the production of the inv and rovA transcripts about 3-fold. We conclude that RNAP can initiate considerable basic transcription starting at the inv and both rovA promoter(s) without RovA, but the RovA protein further activates inv and rovA transcription directly.

In contrast to these results, a relative low basal activity of the inv and rovA promoter(s) was observed in vivo (7, 8). This may be explained by the fact that the promoters are all subject to silencing by H-NS, which is not present in the in vitro system. To confirm this, in vitro transcription experiments with the inv and rovA promoter fragments were performed in the presence of purified H-NS of Y. pseudotuberculosis. The data presented in Fig. 2 demonstrate that inv and rovA transcription from both promoters was strongly reduced in a concentration-dependent manner when H-NS was present, whereas the production of the RNA I control transcript remained unaffected. When both H-NS and the RovA protein were present, significantly higher amounts of the inv and rovA run-off transcripts were observed compared with samples in which only the H-NS protein was applied. Derepression of the P2rovA promoter was less pronounced than that of P1rovA, but the transcription start site seemed more defined. Taken together, these results are in agreement with the action of RovA both as direct transcriptional activator and as antirepressor when the negative regulatory factor H-NS is present.

Interaction of RovA with RNA Polymerase—To further test whether direct activation of transcription occurs via RovA interaction with RNAP, we performed in vitro cross-linking experiments with purified RovA and the RNAP core enzyme of E. coli. Western blot analysis with anti-RovA antibodies demonstrated the formation of higher molecular RovA-core enzyme complexes (Fig. 3A), whereas no complexes were seen in samples without RovA or RNAP. This indicated that RovA is able to interact directly with the RNAP core enzyme in a DNA-independent manner. We also performed gel retardation assays with an inv promoter fragment including a RovA-binding site, RNAP, and/or RovA on ice to prevent open complex formation. A single band of binary RNAP-DNA complexes was observed in the sample without activator (Fig. 3B). RovA-DNA complexes and bands with slightly lower mobility than the RNAP-DNA complex were detected in the samples containing activator. We presume that these species represent the ternary complex. In total, more RNAP-DNA complexes were detectable in the presence of RovA, suggesting that the ternary complex was more stable than the binary complex.



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  		FIGURE 2.
In vitro run-off transcription from the inv and rovA promoters. Plasmids pHT102, pHT103, and pHT104 were incubated with either E. coli RNAP in the presence or absence of increasing concentrations of RovA and H-NS as detailed under "Experimental Procedures." pHT102 carries a 3'-terminally truncated inv gene and generates a transcript of 136 nt (A), pHT103 harbors a 3'-terminally truncated rovA gene and generates a transcript from P2 of 124 nt (B), and pHT104 carries a 3'-terminally truncated rovA gene and leads to a transcript of 115 nt from P1 (C). The inv and rovA transcripts and the RNA I transcripts (106 and 108 nt) encoded by the vector are indicated on the right; a radiolabeled DNA marker fragment of 135 nt was loaded on the left. Numbers below show the quantification of the result, the inv' and rovA' transcripts were normalized to RNA I transcripts.

 
The {alpha} C-terminal domain ({alpha}-CTD) of the RNAP is involved in the response to certain transcriptional activator proteins, i.e. some members of AraC family (27). In this case, overexpression of certain {alpha}-CTD variants allows them to compete with wild-type RpoA for assembly into RNAP holoenzyme. If the variant is altered at a residue important for the response to an activator, this expression results in diminished transcription of the target promoter (28). A plasmid-encoded inv-phoA fusion is expressed in E. coli in a temperature-dependent manner similar to Yersinia in the presence of RovA (7). This allowed us to test the effect of overexpression of a series of alanine substitutions throughout {alpha}-CTD of RNAP from residues 255 and 329 (kindly provided by R. Gourse) on RovA-mediated inv activation in E. coli. As shown in Fig. 3C, expression of the RpoA variants Arg265, Asn268, Lys298, and Ser299 had a negative effect and decreased inv expression by 50-70%. All these residues have previously been shown to be important for transcriptional activation by other regulatory proteins (28, 29), indicating that the {alpha}-CTD of RNAP plays a role in activation of inv and rovA expression by RovA.

Identification of Amino Acids in RovA That Prevent RovA-mediated Activation of the inv Gene—To better understand how RovA activates gene transcription, we identified amino acids that are critical for RovA function. As a first step we randomly mutagenized a cloned copy of the rovA gene (pGN12) and employed a RovA-regulated inv-phoA reporter fusion (pAK002) for assessing the function of RovA in Pinv activation. Ten independent rounds of mutagenesis ({approx}10.000 colonies screened) yielded 31 different independent mutations in the rovA gene, which decreased transcription of the inv promoter more than 50% (data not shown). DNA sequencing of the rovA gene revealed several different groups of mutations. One group caused an early stop of rovA mRNA translation due to the insertion of transposable elements (P61::IS1, A10::IS5, G105::Tn5) or point mutations (W16Opal, Q31Ochre, W34Opal, Q47Ochre, Q60Ochre) introducing a stop codon that probably results in the formation of unstable truncated RovA molecules, which were undetectable with a polyclonal anti-RovA antibody (data not shown). 19 of the isolated mutations were missense mutations that resulted in amino acid substitutions in 14 different residues located in the center or in the terminal regions of the RovA protein.

The Internal Region of RovA (Pro44-Arg78) Is Involved in DNA Binding to the inv Promoter—A total of six different single point mutations (P44L, Q49R, Q51R, Q60R, P61L, E71K) and four double point mutations (P45L/Q51R, Q51R/R65K, Q51R/E71K, Q51R/R78W) located in the internal cluster of the RovA protein. Most mutants were highly defective and completely unable to induce inv expression (Fig. 4A), without significantly influencing the stability of the protein (Fig. 4B). Only one RovA mutant (Q51R) in the central region showed residual activator activity to levels about 45% of that of the RovA wild-type protein. Because the double mutants are significantly less active than the single Q51R mutant, it is suggested that mutations at both residues function cumulatively to lower RovA activity. All exchanged amino acids are conserved among the RovA/SlyA proteins and cluster in a region of 30 residues (Pro44-Arg78) that is most highly conserved in this regulatory family. Although the overall sequence identity is low, this region resembles the helix-turn-helix (HTH) region in the winged-helix motif of the three crystallized MarR-like proteins that is believed to mediate DNA interactions (supplemental Fig. A) (19-21). For this reason, we began our analysis by testing whether these mutant RovA proteins are impaired in their capacity to bind target DNA. A subset of the rovA mutant alleles was cloned into a His-tag expression vector. The different RovA variants were purified through a Ni-affinity column and were assessed for DNA binding by mobility shift assays using a PCR-generated DNA fragment, harboring a RovA binding site of the inv regulatory region. As a positive control we tested DNA binding with the RovA wild-type protein. As shown in Fig. 4C, wild-type RovA bound to the inv promoter fragment, whereas none of the tested internal RovA mutants (P44L-E71K) was capable of forming a slower migrating higher molecular weight complex. The DNA binding capacity of the P61L mutant was completely impaired, whereas P44L, Q51R, Q60R, and E71K still exhibited very low DNA binding capabilities, which were only detectable when 5-fold higher concentrations of Q51R or more than 10-fold higher concentrations of the other mutants were added (data not shown). Taken together, these data demonstrated that the changes in the Pro44-Arg78 amino acid cluster impaired the interaction of the RovA protein with DNA and confirmed the postulated winged-helix domain as being important for DNA binding.



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  		FIGURE 3.
Interaction of RovA with RNA polymerase. A, in vitro cross-linking of RovA and RNAP core enzyme (RNAP). Purified RNAP and RovA were incubated without (-) or with 0.1% (+) formaldehyde and analyzed by Western blot using anti-RovA antibodies. Arrows indicate the position of the RovA monomer, dimer, and tetramer, and the solid line marks cross-linked wild-type RovA-RNAP complexes on the right; the size and position of molecular weight standard proteins are indicated on the left. B, gel retardation of an inv promoter fragment harboring a RovA binding site with RNAP, RovA, or both (1:1 ratio). The ternary RovA-RNAP-DNA complexes are indicated by a solid line, and the RNAP-DNA and RovA-DNA complexes are marked by arrows. A 100-bp DNA marker is loaded on the left, and the marker sizes are indicated. C, effects of single alanine substitutions at positions 255 to 329 in {alpha}-CTD on RovA mediated inv transcription. E. coli strains expressing an inv-phoA fusion and the rovA wild-type (wt) gene contained plasmids encoding either wt rpoA or mutant rpoA alleles (pHTf1 and pREII derivatives). Values of alkaline phosphatase activity were averaged from three different experiments and are graphed as percentages of the wild-type (wt) RpoA value. Error bars represent standard deviations. For clarity, only a representative selection of the mutants is shown, positions where Ala is the native residue were not analyzed.

 
To further investigate the DNA binding mutants, we coexpressed the RovA mutants (Q49R, Q60R, or P61L) and the RovA wild-type protein. To do so, Y. pseudotuberculosis strain YP1 (rovA+), harboring a chromosomal inv-phoA indicator fusion, was transformed with the mutant plasmids, and the effect on inv-phoA activation was measured. As a control the effect of plasmids harboring the rovA wild-type plasmid or the rovA stop mutation (Q47Ochre) were also analyzed in YP1. As shown in TABLE ONE, the Q47Ochre mutation was recessive to the presence of the RovA wild-type protein, whereas the RovA mutants Q49R, Q60R, and P61L exerted a trans-acting dominant negative phenotype on RovA-dependent inv activation. They reduced expression of the chromosomal inv-phoA fusion to ~25-70%, most probably as a result of direct association with wild-type RovA or due to competition for interaction with RNAP. Additional cross-linking experiments with all the DNA binding mutants revealed dimeric cross-linking products identical to wild-type RovA, (supplemental Fig. C), demonstrating that substitutions in the putative HTH DNA-binding region were not impaired in RovA-RovA interaction. Futhermore, we used circular dichroism spectroscopy to investigate whether the mutations, in particular the P61L exchange, caused folding defects in the HTH motif, leading to the failure to bind DNA. However, no apparent alterations in the protein conformation were detectable (data not shown).


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  		TABLE ONE
Influence of RovA mutants with internal amino acid substitutions on inv expression in a rovA+ strain

Y. pseudotuberculosis strain YP1 harboring a chromosomally encoded inv-phoA fusion and a plasmid-encoded rovA wild-type copy or various rovA mutant genes were grown at 25 °C in LB medium. Alkaline phosphatase activity was determined from overnight cultures and given in µmol min–1 mg–1. The data represent the average ± S.D. from three different experiments each done in duplicates.

 
The N-terminal Region of RovA Is Essential for Protein Stability and Important for DNA Binding—During the course of mutagenesis three additional changes with a strong effect on the capacity of RovA to activate inv transcription were identified in the N-terminal part of RovA (A10T, L12A, W16A), indicating that this region of the regulatory protein is also critical for its function (Fig. 4A). To further study this portion of the protein, we constructed N-terminal deleted rovA alleles. Only low amounts of the shortest RovA deletion product {Delta}2-8, but not {Delta}2-17 or shorter variants, were detectable in whole cell extracts (Fig. 5B), indicating that the N terminus of the RovA protein is crucial for RovA stability. Similar to the single amino acid substitutions in this region, the RovA{Delta}2-8 deletion variant was unable to activate the inv gene (Fig. 5A), even when its expression was artificially induced and raised to wild-type level. Subsequently, purified His-tagged versions of the different non-active RovA variants were used to determine DNA binding and dimer formation as described. All these mutants retained their ability to form dimers (supplemental Fig. C) but exhibited a significantly reduced ability to interact with DNA. The missense mutants L12A and W16A did not interact with the inv promoter fragment. RovA variants A10T and {Delta}2-8 still bound to DNA but only when higher protein concentrations were used (Figs. 4C and 5C).



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  		FIGURE 4.
Analysis of amino acids substitutions in RovA that render it defective for transcriptional activation. A, E. coli K-12 expressing an invphoA fusion were transformed with the mutagenized rovA+ plasmid, and alkaline phosphatase activity of the cultures was determined. V, pBAD33 vector; wt, pGN12 rovA+ plasmid. The data represent the average ± S.D. from three independent cultures done in duplicate and are presented in µmol min-1 mg-1. B, immunoblotting of E. coli expressing RovA or the different RovA mutant derivatives with an RovA-specific antibody. The RovA proteins are marked by arrows; the position of standard proteins is indicated on the left. C, interaction of the RovA mutant proteins with a RovA-binding site encoded by an inv promoter fragment. Equal amounts of the fragment (0.1 µg) were incubated with (0.06 µg) or without (-) the different RovA proteins in a 20-µl reaction mix. The DNA-RovA complexes were separated on a 4% polyacrylamide gel; the positions of the higher molecular weight RovA-DNA complexes formed are shown by arrows. The molecular weights of the 100-bp ladder marker are indicated on the left. The proteins used are indicated beneath the gel.

 
The C-terminal Region of the RovA Protein Is Involved in Oligomerization and Activation of Gene Transcription—Two additional spontaneous changes in the C-terminal part of RovA (E122K, Q141Ochre) were identified, which strongly affected RovA-mediated inv expression (Figs. 4B and 5B). Further studies with the purified E122K mutant demonstrated that this RovA variant retained its ability to form dimers, even though the number of dimers was significantly reduced (supplemental Fig. C). E122K was still capable to interact with the inv promoter fragment harboring the RovA binding site, although a smaller RovA-DNA nucleoprotein complex was detected in mobility shift assays (Fig. 4C). Formation of an additional nucleoprotein band of the size of the wildtype RovA-DNA complex could only be detected under higher protein concentrations (supplemental Fig. D). DNase I footprinting analysis further demonstrated that low amounts of E122K interacted specifically with one half-site of the RovA binding region. Only when more protein was added, the other half-site also became occupied by the E122K protein, whereas the entire site was bound by the RovA wild-type protein under low protein concentrations (supplemental Fig. D). This result further indicated that the capability of E122K to form oligomers is reduced.



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  		FIGURE 5.
Analysis of RovA N- and C-terminal deletion mutants that render it defective for transcriptional activation. A, E. coli strain CC118{lambda}pir pAK002 expressing an inv-phoA fusion and the RovA wt or one of the mutant derivatives were grown overnight in LB, and alkaline phosphatase activity of the cultures was determined. The vector control (V) was pBAD33. The data represent the average ± S.D. from three independent cultures done in duplicate and are presented in µmol min-1 mg-1. B, 1 ml from these cultures were pelleted and resuspended in a volume of sample buffer so as to normalize for A600. Equivalent amounts of the cell extracts were separated on a 15% SDS-polyacrylamide gel and then analyzed by Western blot using an anti-RovA antibody. The RovA proteins are marked by arrows; the size and position of a molecular weight standard protein are indicated on the left. C, interaction of the RovA mutant proteins with DNA. An individual inv promoter fragment harboring a RovA-binding site was incubated without (-) or with increasing amounts of the different RovA proteins. The DNA-RovA complexes were separated on a 4% poly-acrylamide gel. A molecular weight standard was loaded on the left, and the corresponding molecular weights are indicated. The proteins used are indicated beneath the gel. The positions of the higher molecular weight RovA-DNA complexes formed are shown by arrows. D, in vitro cross-linking of the different RovA deletion mutants. The purified RovA proteins were incubated without (-) or with (+) 12 mM AMAS for 2 h at room temperature and the monomeric (1-mer) and cross-linked dimeric (2-mer) products were detected as described in the legend to Fig. 1A.

 
To investigate the role of the C-terminal amino acids of RovA in more detail, we also introduced alanine substitutions into residues (Ser119, Leu129, Lys132), which are mostly conserved among the MarR-family (supplemental Fig. A), and constructed an additional set of C-terminal deletions. Unlike E122K none of the missense mutations had a severe effect on RovA activity (Fig. 4A). In contrast, all C-terminal deletions permitted the synthesis of stable RovA proteins, which exhibited a significant lower ability to activate inv expression (Fig. 5A). Even the loss of the most extreme C-terminal amino acid lysine led to a reduction of transcriptional activation of about 35%. Progressive deletions of additional amino acids successively decreased the transcriptional activity of RovA and C-terminal deletions of four and more amino acids fully eliminated RovA-mediated induction of inv expression. This demonstrated that the extreme C-terminal amino acids of RovA are crucial for RovA function. As two different substitutions (K143Q, K143E) of the single charged amino acid Lys143 in this region had no or only a slight effect on RovA-mediated transcription activation (Fig. 5A), it appeared that the charge is not critical for RovA-mediated gene expression. To further elucidate the defects of the non-active C-terminal RovA mutants, we used purified His-tagged versions of the RovA deletions C108*, D121*, and L140* to determine DNA binding and dimer formation as described. We found that the C108* and D121* proteins were not or much less able to dimerize and did not interact with the inv promoter fragment, even when 10-fold higher amounts of the purified proteins were added (Fig. 5, C and D). This supports our previous results, indicating that internal parts of the C terminus are implicated in oligomerization of RovA.



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  		FIGURE 6.
In vitro run-off transcription from the inv promoter in the presence of the RovA wt protein, RovA L140*, and/or H-NS. Plasmid pHT102 was incubated with E. coli RNAP and increasing concentrations of RovA, L140*, and/or H-NS as detailed under "Experimental Procedures." pHT102 carries a 3'-terminally truncated inv gene and generates a transcript of 136 nucleotides. The inv and rovA transcripts and the RNA I transcripts (106 and 108 nt) encoded by the vector are indicated by arrows. A radiolabeled DNA marker fragment of 135 nt is indicated on the left. Numbers below show the quantification of the result (relative transcription), the inv' transcripts were normalized to RNA I transcripts.

 
The RovA deletion variant L140*, just missing the last four C-terminal amino acids, was also unable to activate an inv-phoA fusion in vivo but fully retained its oligomerization and DNA binding function (Fig. 5). No changes in L140*-DNA complex formation were detectable, and no clear loss of DNA binding affinity to the inv promoter fragment was observed in gel retardation or DNase I footprinting assays with serial dilutions of RovA and L140* (Fig. 5C and supplemental Fig. D). Presumably, lack of L140* function results from small structural alterations, which may affect activation of the transcriptional machinery.

The L140* Mutant Is Reduced in Direct Activation and Fully Defective in Replacing H-NS—To further address whether direct activation of the inv promoter is impaired in the L140* protein we performed in vitro transcription assays with purified L140* and compared its activity with that of the RovA wild-type protein. Although expression of His-tagged L140* did not induce an inv-phoA fusion in vivo (data not shown), we found that this protein was still able to induce transcription starting from the RovA-dependent Pinv promoter. However, the efficiency of activation was reduced compared with the RovA wild-type protein (Fig. 6A). Complex formation with RNAP was still detectable in cross-linking experiments (data not shown), indicating that the loss of amino acids 140-143 did not fully abrogate the interaction of RovA with RNA polymerase. Furthermore, we found that clearance of the inv promoter by the replacement of H-NS seemed severely impaired with the RovA L140* protein. As shown in Fig. 6B, efficient transcription of the inv promoter with RovA wild-type protein occurred in the presence of H-NS, whereas no or much less inv specific transcripts were detected when the L140* mutant protein was applied.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of virulence factors is essential for the successful establishment of pathogenic bacteria in the host. Virulence gene regulation involves the concerted action of multiple regulatory proteins that bind to DNA, to each other, and interact with the transcriptional machinery. Such regulation provides a way to respond to physiological signals, such as those that initiate the expression of virulence genes. In this study we analyzed the functional properties of RovA, an uncharacterized transcriptional factor that belongs to the SlyA/Hor subfamily of MarR-like regulatory proteins. RovA plays an important role in controlling virulence gene expression in enteropathogenic Yersinia spp (6, 7). RovA activates the expression of the primary internalization factor invasin and its own gene (8). In this capacity, the function of RovA is different from that of MarR and other MarR-like proteins, which typically act as transcriptional represssors. This report describes in vitro transcription studies and mutational analysis of the RovA protein aimed to characterize the requirements for RovA to activate transcription at the inv and rovA promoters. The data presented here lead us to conclude that transcriptional activation by the RovA/SlyA/Hor subfamily occurs directly through interaction with RNAP and indirectly through replacement of the histone-like protein H-NS, a feature that has not yet been attributed to other MarR-like proteins, and we first defined the structural domains of this regulator family that contribute to activation function.

In vitro transcription reactions in this study demonstrate that RNAP alone induces transcription of the inv and the two rovA promoters to some degree. Addition of the RovA protein clearly enhanced the level of all transcripts generated, showing that RovA is a direct activator which stimulates RNAP without accessory factors. In our previous study we demonstrated that the DNA binding sites of RovA in the inv and the rovA regulatory region are positioned upstream of the -35 element of the promoters (8). This arrangement is characteristic for most class I activator-dependent promoters, and their activation usually requires interactions of the DNA-bound activator protein with the {alpha}-CTD of RNAP. This enhances the formation and stability of the initial RNAP-promoter complex and may affect the rate of subsequent conformational changes required to form the open complex (30). It is also possible that RovA makes protein-protein contacts with RNAP in solution prior to DNA binding or during their diffusion along DNA as proposed for other activator proteins (31, 32). The identification of RovA-RNAP complexes in this study and the observation of a dominant negative effect of certain RpoA variants on RovA-mediated inv expression (Fig. 3) support a role of these mechanisms in RovA-mediated gene activation. Four alanine substitutions in the RpoA residues Arg265, Asn268, Lys298, and Ser299 strongly reduce RovA-mediated inv expression. All these residues were previously shown to be important for transcriptional activation by other {alpha}-CTD dependent activator proteins, such as CRP and AraC. They localize in a surface-exposed complex face that is involved in nonspecific protein-DNA interactions and stabilize the activator-{alpha} subunit contact (28, 33).



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  		FIGURE 7.
Model of the RovA dimer structure. RovA homology modeling was performed with SWISS-MODEL (38), using the crystal structure of the MarR-like protein MexR of similar length as template. The sequences were aligned using the 3D-PSSM server (39), and the reliability of the model was varified with the programs WHAT-CHECK and ProsaII (40, 41), both indicating a good overall agreement with experimental data. The two RovA monomers are colored red and blue. Significant residues on RovA, which correspond to unfunctional amino acid substitution, are indicated and shown in stick rendering. The {alpha}-helices of one monomer (red) are indicated and the N and C termini are labeled N and C, respectively.

 
The overall effect of RovA on inv and rovA transcription in vitro is clearly lower compared with the activation of inv-phoA and rovA-lacZ expression in vivo (7, 8). This shows that RovA-dependent activation of gene expression in vivo is more complex and reflects additional effects by other regulatory proteins, e.g. H-NS. In fact, no or significantly less inv and rovA transcripts are produced when the nucleoid-associated protein H-NS is added to the in vitro transcription system. In contrast, H-NS-mediated repression is fully relieved when RovA is present; under these conditions inv and rovA are transcribed at similar or higher levels than with RNAP alone (Fig. 2). This reflects what we previously observed in vivo, in which removal of H-NS led to higher inv and rovA expression levels even in the absence of RovA (8). Presumably, H-NS has a relatively strong effect on inv and rovA transcription because the regulatory region of inv and rovA contains several binding sites for H-NS (8). In summary, transcriptional levels of RovA-dependent genes reflect direct activation of the transcriptional machinery, the outcome of RovA/H-NS competition, and the rovA autoregulatory mechanism. In this respect, RovA resembles virulence gene regulators of the AraC-family, such as ToxT of Vibrio cholerae that controls virulence genes required for the biogenesis of the toxin-co-regulated pilus and cholera toxin and PerA from enteropathogenic E. coli that regulates the expression of the bundle-forming pilus operon and the LEE pathogenicity island (34, 35). In all these cases H-NS silencing may contribute to prevent virulence gene expression until the activator reaches a sufficient level through the autoregulatory loop that controls its expression.

In our attempt to define functional domains of RovA, we identified various missense and deletion mutants of RovA that were impaired in activating inv expression. The characterization of the RovA mutants in this study as well as in silico analysis and modeling indicate that the overall structure of RovA appears to resemble that of MarR and MexR (19), despite their low amino acid sequence similarity. Taken together, this allowed us to propose a structural model for the RovA protein (Fig. 7). Most of our isolated substitutions are clustered in the central region (amino acids 44-78) and are strongly impaired in DNA binding. This internal region exhibits the highest homology to other MarR-like proteins (supplemental Fig. A) and corresponds to the putative DNA binding domain of MarR that adopts a winged-helix fold, consisting of a HTH motif followed by two {beta}-sheets (19). How MarR-like proteins interact with DNA is unknown, but sequence recognition by other winged-helix proteins is generally achieved by interaction of the HTH recognition helix with DNA. In addition, the wings can contact the DNA in the minor groove and/or the phosphate backbone (20, 36, 37). The isolated amino acid changes in the central region of RovA locate in the postulated recognition helix {alpha}4 or in the stabilization helix {alpha}3 of the HTH motif (Fig. 7). This would explain why the DNA binding ability of these mutants is strongly reduced.

Gel filtration and cross-linking experiments in this work show that RovA predominantly forms dimers in solution. From our mutation analysis it appears that the HTH region does not contribute to RovA dimerization, but it is evident that even small changes in both termini of the RovA protein have a severe impact on RovA structure and function. N-terminal deletions are very unstable and amino acid substitutions A10T, L12A, and W16A, which localize within a predicted {alpha}-helical domain (amino acids 4-23), compromised DNA binding and abolished inv activation. Deletions (C108*, D121*) removing the last one or two predicted helices {alpha}6 and {alpha}5 or the substitution E122K in this region, fully abrogated RovA function, affected DNA binding, and strongly reduced the capacity to form dimers. Thus, it is most likely that these domains play a structural role, helping to stabilize the formation of dimers with properly positioned DNA-binding segments. The dimerization domain of the three crystallized MarR-like proteins all include hydrophobic interactions between helices {alpha}1, {alpha}5, and {alpha}6 that are stabilized by diverse intermolecular bonds. The first N-terminal helix {alpha}1 was shown to be inserted between the last C-terminal helices {alpha}5 and {alpha}6, which all contribute to the formation of an extensive and well packed dimer interface (19-21). According to our mutation analysis, it is very likely that the terminal regions of RovA fulfil a similar function in dimer formation (Fig. 7).

Although the DNA binding mechanism and dimerization of the activator RovA and the repressor proteins MarR and MexR appear to be similar, RovA possesses additional functions and properties that apparently allow RovA to specifically activate virulence gene expression. We showed that the C-terminal tail of the RovA protein, which is missing or very different in other MarR-like proteins, is important for gene activation. Successive deletions of the final residues result in a continuous loss of inv promoter activation and a deletion of the last four amino acids (L140*) is unable to activate an inv-phoA fusion in vivo. The L140* protein forms dimers and specifically interacts with the inv promoter region, suggesting that the overall conformation is unchanged and the binding to its DNA target site is essentially intact. However, the capacity of L140* to activate transcription is reduced and the ability of L140* to displace H-NS is strongly impaired, if not abolished. According to structure predictions the C-terminal tail of the RovA protein is surface-exposed (Fig. 7) and could make contacts with other amino acid side chains. This might stabilize RovA interaction with RNAP and/or could manipulate the function of other regulatory proteins such as H-NS. It is also possible that the final amino acids mediate interdimer contacts when RovA is bound to adjacent binding sites upstream the inv and rovA promoters (8), and this may be important to displace H-NS and activate transcription. Further structural studies of RovA in the presence of DNA are required to fully understand how the C-terminal end of RovA contributes to trancriptional activation.


   FOOTNOTES
 
* This work was supported by Grant DE 616/3 from the Deutsche Forschungsgemeinschaft (to P. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A-D. Back

1 To whom correspondence should be addressed. Tel.: 49-30-4547-2373; Fax: 49-30-4547-2328; E-mail: derschp{at}rki.de.

2 The abbreviations used are: nt, nucleotide(s); RNAP, RNA polymerase; {alpha}-CTD, {alpha} C-terminal domain; HTH, helix-turn-helix; wt, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Martin Fenner, Abigail Mavor, and Barbara Weissenmayer for helpful discussions and critical reading of the manuscript. We also thank Richard Gourse for plasmids and Athanasios Typas for excellent advice concerning the in vitro transcription experiments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bottone, E. J. (1997) Clin Microbiol. Rev. 10, 257-276[Abstract]
  2. Smego, R. A., Frean, J., and Koornhof, H. J. (1999) Eur. J. Clin. Microbiol. Infect. Dis. 18, 1-15[CrossRef][Medline] [Order article via Infotrieve]
  3. Grutzkau, A., Hanski, C., Hahn, H., and Riecken, E. O. (1990) Gut 31, 1011-1015[Abstract/Free Full Text]
  4. Marra, A., and Isberg, R. R. (1997) Infect. Immun. 65, 3412-3421[Abstract]
  5. Pepe, J. C., and Miller, V. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6473-6477[Abstract/Free Full Text]
  6. Revell, P. A., and Miller, V. L. (2000) Mol. Microbiol. 35, 677-685[CrossRef][Medline] [Order article via Infotrieve]
  7. Nagel, G., Lahrz, A., and Dersch, P. (2001) Mol. Microbiol. 41, 1249-1269[CrossRef][Medline] [Order article via Infotrieve]
  8. Heroven, A. K., Nagel, G., Tran, H. J., Parr, S., and Dersch, P. (2004) Mol. Microbiol. 53, 871-888[CrossRef][Medline] [Order article via Infotrieve]
  9. Dube, P. H., Revell, P. A., Chaplin, D. D., Lorenz, R. G., and Miller, V. L. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 10880-10885[Abstract/Free Full Text]
  10. Daniels, J. J., Autenrieth, I. B., Ludwig, A., and Goebel, W. (1996) Infect. Immun. 64, 5075-5084[Abstract]
  11. Buchmeier, N., Bossie, S., Chen, C. Y., Fang, F. C., Guiney, D. G., and Libby, S. J. (1997) Infect. Immun. 65, 3725-3730[Abstract]
  12. Libby, S. J., Goebel, W., Ludwig, A., Buchmeier, N., Bowe, F., Fang, F. C., Guiney, D. G., Songer, J. G., and Heffron, F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 489-493[Abstract/Free Full Text]
  13. Spory, A., Bosserhoff, A., von Rhein, C., Goebel, W., and Ludwig, A. (2002) J. Bacteriol. 184, 3549-3559[Abstract/Free Full Text]
  14. Oscarsson, J., Mizunoe, Y., Uhlin, B. E., and Haydon, D. J. (1996) Mol. Microbiol. 20, 191-199[Medline] [Order article via Infotrieve]
  15. Ludwig, A., Bauer, S., Benz, R., Bergmann, B., and Goebel, W. (1999) Mol. Microbiol. 31, 557-567[CrossRef][Medline] [Order article via Infotrieve]
  16. Thomson, N. R., Cox, A., Bycroft, B. W., Stewart, G. S., Williams, P., and Salmond, G. P. (1997) Mol. Microbiol. 26, 531-544[CrossRef][Medline] [Order article via Infotrieve]
  17. Alekshun, M. N., and Levy, S. B. (1999) J. Bacteriol. 181, 4669-4672[Abstract/Free Full Text]
  18. del Castillo, I., Gómez, J. M., and Moreno, F. (1990) J. Bacteriol. 172, 437-445[Abstract/Free Full Text]
  19. Alekshun, M. N., Levy, S. B., Mealy, T. R., Seaton, B. A., and Head, J. F. (2001) Nat. Struct. Biol. 8, 710-714[CrossRef][Medline] [Order article via Infotrieve]
  20. Lim, D., Poole, K., and Strynadka, N. C. (2002) J. Biol. Chem. 277, 29253-29259[Abstract/Free Full Text]
  21. Wu, R. Y., Zhang, R. G., Zagnitko, O., Dementieva, I., Maltzev, N., Watson, J. D., Laskowski, R., Gornicki, P., and Joachimiak, A. (2003) J. Biol. Chem. 278, 20240-20244[Abstract/Free Full Text]
  22. Heroven, A. K., and Dersch, P. (2002) Trends Microbiol. 10, 267-268[CrossRef][Medline] [Order article via Infotrieve]
  23. Ferrandiz, M. J., Bishop, K., Williams, P., and Withers, H. (2005) Infect. Immun. 73, 1684-1694[Abstract/Free Full Text]
  24. Sambrook, J. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  25. Miller, J. H. (1992) A Short Course in Bacterial Genetic: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Laboratories, C. S. H., ed) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  26. Miller, J. H. (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  27. Gallegos, M. T., Schleif, R., Bairoch, A., Hofmann, K., and Ramos, J. L. (1997) Microbiol. Mol. Biol. Rev. 61, 393-410[Abstract]
  28. Gaal, T., Ross, W., Blatter, E. E., Tang, H., Jia, X., Krishnan, V. V., Assa-Munt, N., Ebright, R. H., and Gourse, R. L. (1996) Genes Dev. 10, 16-26[Abstract/Free Full Text]
  29. Murakami, K., Fujita, N., and Ishihama, A. (1996) EMBO J. 15, 4358-4367[Medline] [Order article via Infotrieve]
  30. Barnard, A., Wolfe, A., and Busby, S. (2004) Curr. Opin. Microbiol. 7, 102-108[CrossRef][Medline] [Order article via Infotrieve]
  31. Griffith, K. L., Shah, I. M., Myers, T. E., O'Neill, M. C., and Wolf, R. E., Jr. (2002) Biochem. Biophys. Res. Commun. 291, 979-986[CrossRef][Medline] [Order article via Infotrieve]
  32. Martin, R. G., Gillette, W. K., Martin, N. I., and Rosner, J. L. (2002) Mol. Microbiol. 43, 355-370[CrossRef][Medline] [Order article via Infotrieve]
  33. Jeon, Y. H., Negishi, T., Shirakawa, M., Yamazaki, T., Fujita, N., Ishihama, A., and Kyogoku, Y. (1995) Science 270, 1495-1497[Abstract/Free Full Text]
  34. Yu, R. R., and DiRita, V. J. (2002) Mol. Microbiol. 43, 119-134[CrossRef][Medline] [Order article via Infotrieve]
  35. Porter, M. E., Mitchell, P., Roe, A. J., Free, A., Smith, D. G., and Gally, D. L. (2004) Mol. Microbiol. 54, 1117-1133[CrossRef][Medline] [Order article via Infotrieve]
  36. Li, R., Manna, A. C., Dai, S., Cheung, A. L., and Zhang, G. (2003) J. Bacteriol. 185, 4219-4225[Abstract/Free Full Text]
  37. Huffman, J. L., and Brennan, R. G. (2002) Curr. Opin. Struct. Biol. 12, 98-106[CrossRef][Medline] [Order article via Infotrieve]
  38. Schwede, T., Kopp, J., Guex, N., and Peitsch, M. (2003) Nuc. Acids Res. 31, 3381-3385[Abstract/Free Full Text]
  39. Kelley, L., MacCallum, R., and Sternberg, M. (2000) J. Mol. Biol. 299, 499-520[Medline] [Order article via Infotrieve]
  40. Hooft, R. W. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve]
  41. Sippl, M. (1993) Proteins 17, 355-362[CrossRef][Medline] [Order article via Infotrieve]

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
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