In vitro binding of the response regulator CitB and of its carboxy-terminal domain to A+T-rich DNA target sequences in the control region of the divergent citC and citS operons of Klebsiella pneumoniae¹

Abstract

The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB. Northern blot analyses described here and elsewhere indicate that each group forms an operon. The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A+T content of 67%. Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator). As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties. To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)₆-tag, were purified. CitB_His and CitBN_His could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC. The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein. Upon phosphorylation, CitB_His became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure. In gel retardation assays, CitB_His bound specifically to the citC-citS intergenic region. The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold. Depending on the protein concentration, four different phospho-CitB_His-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS. DNase I footprints revealed two protected regions extending maximally from −55 to −89 relative to the citS transcription start and from −50 to −96 relative to the citC transcription start. Gel retardation and DNase I footprint assays with CitBC_His showed that the C-terminal domain is sufficient for specific DNA binding. Since its properties were similar to that of unphosphorylated CitB_His, an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated. The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase.

Introduction

Klebsiella pneumoniae is able to grow anaerobically with citrate as sole source of carbon and energy, and forms acetate, formate and CO₂ as endproducts (for reviews, see Bott 1997, Dimroth 1997). Three enzymes are specifically required for this type of fermentation: a Na⁺-dependent citrate carrier Pos et al 1994, Pos and Dimroth 1996, citrate lyase (Bott & Dimroth, 1994), and the Na⁺ pump oxaloacetate decarboxylase (Di Berardino & Dimroth, 1996). These proteins are induced under anoxic conditions in the presence of citrate and Na⁺ and are subject to catabolite repression Bott et al 1995, Dagley and Dawes 1953, O’Brien 1975a, O’Brien 1975b. Analysis of the corresponding genes revealed that they form two divergently transcribed clusters on the K. pneumoniae chromosome, which are separated by a small intergenic region (Figure 1). The citS-oadGAB cluster encodes the citrate carrier and the three subunits of oxaloacetate decarboxylase van der Rest et al 1992, Schwarz and Oesterhelt 1985, Schwarz et al 1988, Laußermair et al 1989, Woehlke et al 1992. The citCDEFG cluster (Bott & Dimroth, 1994) encodes a ligase (CitC) catalysing the ATP-dependent acetylation of the phosphoribosyl dephospho-coenzyme A prosthetic group of citrate lyase, the three different subunits of citrate lyase (CitDEF), and a protein of yet unknown function (CitG). Due to the physical proximity to citF and its conservation in the citCDEFG gene cluster from Haemophilus influenzae(Fleischmann et al., 1995), citG might encode a protein involved in the conversion of apo-citrate lyase to holo-citrate lyase. Downstream of citG the gene encoding dihydrodipicolinate reductase (dapB) was identified, an enzyme catalysing a step in the biosynthesis of lysine (Bott & Dimroth, 1994).

Recently, we started an approach to characterize the cis and trans-acting elements involved in the regulation of the citrate-specific fermentation genes (Bott et al., 1995). In search of genes required for expression of citS from its native promoter in Escherichia coli, a two-component signal transduction system was identified consisting of the sensor kinase CitA (61.8 kDa) and the response regulator CitB (26.8 kDa). Analysis of the primary structure of CitA indicated that the protein is membrane-bound and consists of three domains: an amino-terminal periplasmic domain, presumably involved in signal recognition, which was flanked by two transmembrane helices, a central cytoplasmic domain of unknown function, and a carboxy-terminal kinase domain containing all of the conserved sequence motifs characteristic of the sensor kinase protein family Parkinson and Kofoid 1992, Stock et al 1995. The response regulator CitB, like many other members of this family, was composed of an amino-terminal receiver domain and a carboxy-terminal output domain with a helix-turn-helix motif typical of DNA-binding proteins. Remarkably, the only protein in the Data Base that exhibits a high degree of identity (46%) with the C-terminal domain of CitB is the response regulator CriR from Shigella flexneri (accession number U29654). This protein has been proposed to act as a transcriptional activator of the ipa genes, whose products are required for the invasion of epithelial cells.

The overlapping genes for citA and citB were located downstream of oadB in the same orientation (Figure 1). K. pneumoniae citB deletion mutants (KM3, KM4), which were constructed by replacing part of the coding region with a kanamycin resistance gene (aph), were unable to grow anaerobically on citrate, because none of the citrate fermentation enzymes, i.e. CitS, citrate lyase, and oxaloacetate decarboxylase, was synthesized any longer. In the case of citA deletion mutants, the phenotype was dependent on the orientation of the inserted aph gene. If aph had an inverse orientation to citA, the citB gene downstream became transcriptionally silent and the resulting mutant (strain KM2) had the same phenotype as the citB deletion mutants. If aph had the same orientation as citA (strain KM1), however, readthrough from the aph promoter led to constitutive high level expression of citB. As a result, the citrate-specific fermentation enzymes were formed constitutively, i.e. independent of citrate, sodium ions, and anoxic conditions (Bott et al., 1995). Studies of the regulation of citAB expression revealed a strong increase of the cellular CitA/CitB levels during anaerobic growth on citrate that was dependent on CitB itself and resulted from co-transcription of citAB with citS-oadGAB(Bott et al., 1995).

The results described above strongly suggested that CitB functions as a transcriptional activator of the genes specifically involved in citrate fermentation. The most obvious target for exertion of this function is the citC-citS intergenic region. Assuming that citS-oadGAB-citAB and citCDEFG form an operon, activation of transcription at the citC and at the citS promoter allows the coordinate induction of all these genes. In the studies described below we confirmed the operon structure of citCDEFG, determined the transcriptional start sites of citS and citC, and identified multiple CitB binding sites in the citC-citS intergenic region using gel retardation and DNase I footprint assays.