| The hex regulon of Pseudomonas aeruginosa, a coordinately regulated multioperonic group of genes, is induced during growth on carbohydrates. The hex regulon encodes the Entner- Doudoroff pathway enzymes, 6-phosphogluconate dehydratase (Edd) and 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase (Eda), as well as glucokinase (Glk), glucose 6-phosphate dehydrogenase (Zwf), 6-phosphogluconolactonase (Pgl), and NAD-specific glyceraldehyde-3- phosphate dehydrogenase (NAD-Gap). Previous studies have shown that the hex regulon is subject to catabolite repress ion control (CRC) mediated by the crc gene product and that a cis-acting region of P. aeruginosa chromosomal DNA, the hexC locus, can titrate a putative regulatory protein when present on a multicopy plasmid To date, however, no specific regulatory proteins have been identified which exert direct control over hex regulon gene expression. Early phases of this study identified at least two potential trans-acting regulatory proteins which bind the hexC locus and could potentially control hex regulon gene expression. One of these putative regulators was integration host factor (IHF), a well characterized procaryotic DNA binding/bending protein. The second protein was designated HexR. P. aeruginosa IHF and HexR were partially purified and are biochemically separable. Multiple putative IHF and HexR. binding sites were identified within the hexC locus and potential promoter regions of zwf. The Eda metabolite KDPG prevents in vitro binding of HexR to the hexC locus and zwf promoter region. Collectively, these results suggest that HexR is a negative regulatory protein that controls hex regulon gene expression. The gene encoding P. aeruginosa hexR was cloned and localized to a 1.5 kb region of P. aeruginosa PAO1 chromosomal DNA by monitoring heterologous expression of HexR DNA-binding activity in Escherichia coli. Nucleotide sequence analysis identified an open-reading frame (ORF) of 858 nucleotides separated from the divergently oriented zwf gene by a 186 bp intergenic region. hexR encodes a protein with a predicted monomeric MW of 31.2 kDa. HexR exhibits >56% identity at the amino acid level to hypothetical regulatory proteins from E. coli, Yersinia pestis and Vibrio cholerae. Disruption of hexR eliminated HexR binding activity in crude extracts of P. aeruginosa and led to constitutive expression of all hex regulon genes. Recombinant HexR was purified to homogeneity. Two HexR binding sites were identified by DNase I footprinting in both the hexC locus and zwf promoter region. Together, these data demonstrate that HexR, a member of a novel class of procaryotic regulatory proteins, is a repressor protein required for controlling the expression of the hex regulon in the absence of inducing carbohydrate and under catabolite-repressing conditions in P. aeruginosa. |
