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7 associated with phosphorylation leads to a -50-fold higher DNA affinity. The C-terminal subdomain of CitB (CitBCHis) was capable of specific binding to the citC-citS intergenic region. The binding properties were comparable with the ones of unphosphorylated CitBHis, indicating that the N-terminal domain is essential for high-affinity DNA binding. Two CitB binding sites within the citC-citS intergenic region were identified by DNase I footprint analysis. One binding site is located in the region of -50 to -96 with respect to the citC transcriptional start site, while the second site protects the region from -55 to -89 upstream of the citS transcriptional start site. The positions of these binding sites imply that transcriptional activation of the citrate fermentation genes could involve a direct interaction of the response regulator CitB with the C-terminal domain of the RNA polymerase c-subunit, Early experiments had indicated that alternative growth substrates might have an influence on the expression of the citrate fermentation genes. Therefore, the impact of glucose, gluconate, and glycerol on growth and expression of a chromosomally integrated translational citS-lacZ fusion was examined. All of the above mentioned substrates led to inhibition of citS-lacZ expression. Anaerobic cultivation on citrate and glucose resulted in diauxic growth. The cAMP receptor protein (CRP) or catabolite activator protein (CAP) is known to be involved in the creation of this phenomenon. Indeed, the comparison of the citC-citS intergenic region with the consensus binding sequence led to the identification of three putative CRP binding sites. CRP from K. pneumoniae was overproduced in E. coli and purified. Gel retardation experiments with CRP and the citC-citS intergenic region in the presence of cAMP revealed two protein-DNA complexes of different mobility. DNase I footprints with CRP in the presence of phosphorylated CitBHis revealed hypersensitive sites that are probably due to CRP-induced bending of the DNA. These hypersensitive sites were mapped to the two postulated CRP binding sites that are localized at a distance of 41.5 bp from the transcriptional start site of citC and citS, respectively. These results indicate, that besides the main regulator CitB also CRP is able to enhance the expression of the citCand the citS-operon.

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Regulation of the citrate fermentation genes in Klebsiella pneumoniae: functional analysis of the two-component system CitA/CitB and of the cAMP receptor protein