Two genes for carbohydrate catabolism are divergently transcribed from a region of DNA containing the hexC locus in Pseudomonas aeruginosa PAO1

Abstract

The hexC locus ofPseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two-to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60%Yo identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobiis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.Pseudomonads utilize a number of alternative pathways for the catabolism of carbohydrates to carbon and energy (reviewed in reference 37). In Pseudomonas aeruginosa, the coordinately regulated enzymes glucokinase (GLK), glucose-6-phosphate dehydrogenase (ZWF), and the Entner-Doudoroff pathway enzymes 6-phosphogluconate dehydratase (EDD) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) play a central role in the dissimilation of glucose to pyruvate and glyceraldehyde-3-phosphate (Fig. 1). These enzymes are coinduced by growth of P. aeruginosa on such diverse carbohydrates as glucose, gluconate, mannitol, and glycerol (33, 37).Also, their expression is subject to catabolite repression control (CRC) by succinate or other intermediates of the tricarboxylic acid (TCA) cycle (40,58).The genes encoding these four enzymes (glk, zwf, edd, and eda) were mapped by transductional analysis to 39 min on the PAO1 chromosome (14,30,47). Cloning and complementation analyses revealed, however, that the four genes were expressed from separate promoters (15,57), indicating that the genes were not contained in a single operon. Two regulatory effects further defined this group of genes. The first was the hexR mutation, thus far inseparable from edd,...

Dual regulation of zwf-1 by both 2-keto-3-deoxy-6-phosphogluconate and oxidative stress in Pseudomonas putida

“…In P. aeruginosa PAO1, a locus designated hexC (129 bp, cis-element) exists between the gap and edd genes (Temple et al, 1994). This region reportedly contains binding sites not only for HexR but also for the integration-host factor (IHF; Proctor et al, 1997).…”

“…( Fig. 1; del Castillo et al, 2007b;Temple et al, 1994). The first of these pathways is the glucose kinase pathway, in which glucose is transported into the cytoplasm by the glucose transport system.…”

“…HexR regulates two operons containing the zwf (glucose-6-phosphate dehydrogenase; G6PDH), pgl (6-phophogluconolactonase) and eda genes; and the edd, glk (glucokinase) and gltR-2 (DNA binding response regulator GltR) genes (del Castillo et al, 2008;Hager et al, 2000;Petruschka et al, 2002;Temple et al, 1994). HexR also regulates the expression of glyceraldehyde-3-phosphate dehydrogenase, encoded by the gap gene (del Castillo et al, 2008), and controls zwf in the ED pathway of Pseudomonas aeruginosa (Hager et al, 2000).…”

Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae

“…All bacterial species use pathways to reduce NAD + to NADH, for example respiration, which involves either glycolysis and the tricarboxylic acid cycle or fermentation (Jurtshuk, 1996). Bacteria also use NAD + in several forms of dehydrogenases when breaking down aldehydes and alcohols (Kotrbova-Kozak et al, 2007;Luong et al, 2015;Nobelmann & Lengeler, 1996;Temple et al, 1994). NAD is utilized in a number of cellular processes in both bacterial and mammalian cells, for example DNA ligation and repair, redox recycling in the pyruvate dehydrogenase pathway, and synthesis of acetyl-CoA for the tricarboxylic acid cycle (Chalkiadaki & Guarente, 2012;Chiarugi et al, 2012;Ishino et al, 1986;Patel et al, 2014;Satoh & Lindahl, 1992;Wilkinson et al, 2001).…”

Intracellular 2-keto-3-deoxy-6-phosphogluconate is the signal for carbon catabolite repression of phenylacetic acid metabolism in Pseudomonas putida KT2440

“…CCR of toluene metabolism by glucose is mediated by 2-keto-3-deoxy-6-phosphogluconate (KDPG), an intermediate in the Entner-Doudoroff (ED) pathway of glucose metabolism in P. putida Velázquez et al, 2004). Interestingly, the regulation of glucose metabolism is controlled by the HexR repressor (Hager et al, 2000;Petruschka et al, 2002;Temple et al, 1994). We have shown that HexR binding to its cognate operator sites is inhibited by KDPG (Kim et al, 2008).…”