Regulation of anaerobic citrate metabolism in Klebsiella pneumoniae

Abstract

Three enzymes are specifically required for uptake and catabolism of citrate by Klebsiella pneumoniae under anaerobic conditions: a Na+ -dependent citrate carrier (CitS), citrate lyase (CitDEF), and the Na+ pump oxaloacetate decarboxylase (OadGAB). The corresponding genes are clustered on the chromosome, with the citCDEFG genes located upstream and divergent to the citS-oadGAB genes. We found that expression of citS from its native promoter in Escherichia coli requires the DNA region downstream of oadB. Nucleotide sequence analysis of this region revealed the presence of two adjacent genes, citA and citB. By sequence similarity, the predicted CitA and CitB proteins were identified as members of the two-component regulatory systems. The sensor kinase CitA contained, in the N-terminal half, two putative transmembrane helices which enclosed a presumably periplasmic domain of about 130 amino acids. The C-terminal half of the response regulator CitB harboured a helix-turn-helix motif typical of DNA-binding proteins. K. pneumoniae citB null mutants were unable to grow anaerobically with citrate as the sole carbon and energy source (Cit- phenotype). When cultivated anaerobically with citrate plus glycerol, all of the citrate-specific fermentation enzymes were synthesized in the wild type, but not in the citB mutants. This showed that citS, oadGAB and citDEF required the CitB protein for expression and therefore are part of a regulon. In the wild type, synthesis of CitS, oxaloacetate decarboxylase and citrate lyase was dependent on the presence of citrate, sodium ions and a low oxygen tension. In a citA null mutant which expressed citB constitutively at high levels, none of these signals was required for the formation of the citrate fermentation enzymes. This result suggested that citrate, Na+, and oxygen exerted their regulatory effects via the CitA/CitB system. In the presence of these signals, the citAB gene products induced their own synthesis. The positive autoregulation occurred via co-transcription of citAB with citS and oadGAB.

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

“…We also found similarity between DpiA and CitB (47% identity, Fig. 2A), the effector protein of a two-component signal transduction system implicated in anaerobic citrate metabolism in Klebsiella pneumoniae (Bott et al, 1995). Immediately 5Ј to dpiA is an open reading frame, named dpiB, that encodes a putative 553-amino-acid peptide showing homology to histidine kinases (modulators) of typical two-component systems (Fig.…”

“…Other amino acids that are conserved among response regulators are shown in italics and a C-terminal helixturn-helix motif (amino acid residues 180-200) potentially involved in DNA binding is underlined (see Fig. 2A, Bott et al, 1995). The dpiA gene was mapped to Kohara clones #167(3G5) and #168(1G6) at 14.1 min on the E. coli chromosome by Southern blot hybridization.…”

“…Amino acid sequence comparison of DpiA to response regulator proteins and DpiB to kinases. The deduced amino acid sequences of DpiA (A) and DpiB (B) were aligned with the Klebsiella pneumoniae homologues CitB and CitA (Bott et al, 1995). Two dots indicate identical amino acids and one dot conserved amino acids.…”

Biosynthesis of the Prosthetic Group of Citrate Lyase

“…The fact that it is not part of the citCDEFG cluster might be accidental, but could also reflect the fact that transcriptional regulation of the citX gene differs from that of the citC operon (21,22). Attempts to clone the K. pneumoniae citX gene by heterologous hybridization with an E. coli citX probe or by PCR with degenerated primers were not successful (data not shown).…”

“…The citC operon is induced under anoxic conditions in the presence of citrate and Na + ions, and its expression is strictly dependent on the CitA/CitB two-component regulatory system (21)(22)(23). Expression of the plasmid-encoded K. pneumoniae citCDEFG genes in Escherichia coli resulted in the formation of an inactive citrate lyase (20).…”