Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa

Abstract

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame ofglpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69%o similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.Mucoid strains of the opportunistic pathogen Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections secrete copious amounts of the extracellular polysaccharide alginate (for comprehensive reviews, see references 17 and 23). Our laboratory is interested in studying the regulated carbohydrate metabolic pathways which are able to provide precursors for alginate biosynthesis. Of particular interest to our studies are the previously reported findings that triose phosphates are obligate intermediates in the biosynthesis of alginate (2) and that fructose 1,6-bisphosphate aldolase is essential for this to occur (3). We are therefore focusing our efforts on one peripheral carbon metabolic pathway which is capable of directly providing these triose phosphate intermediates (3), namely, the glycerol metabolic pathway.In P. aeruginosa, glycerol is primarily metabolized through the Entner-Doudoroff pathway (6,18). Glycerol is transported into the cell by a specific, inducible transport system (glpT). Unlike the case for Escherichia coli, in P. aeruginosa glycerol transport seems to be mediated by a high-affinity, binding protein-dependent transport system (32,37 Table 1. LB medium (24) was used as the rich medium for both E. coli and P. aeruginosa. As minimal medium, BSM medium (6), M9 medium (24), VB medium (38), or LVM medium (13) was used. VBMM medium is VB medium containing 0.3% trisodium citrate. Carbon sources were incorporated into minimal media at a final concentration of 10 mM, and amino acid requirements were satisfied by addition of these components to a final concentration of 0.5 mM. Antibiotics used in selection media were ampicillin at 100 ,ug/ml for E. coli and carbenicillin at 500 ,ug/ml for P. a...

Identification of Pseudomonas aeruginosa glpM, whose gene product is required for efficient alginate biosynthesis from various carbon sources

“…First, we previously described that the potentially strong stem-loop structure that could be formed by the palindromic sequences of the glpD-glpM intergenic region (Fig. 1B) does not function as an efficient transcriptional terminator (29). Second, a cistronic organization of glpD and glpM may explain the polar effects of the ⍀Tc insertion in strain PAO151M (see above).…”

“…Although some of the basic events involved in glycerol metabolism were studied some time ago (4,18,29,31), we have only recently begun to unravel at the molecular level the pathway involved in glycerol metabolism in P. aeruginosa. As a first step in elucidating the molecular organization and mode(s) of regulation of the glp genes of P. aeruginosa, we have recently cloned the glpD gene, encoding sn-glycerol-3-phosphate dehydrogenase, a key enzyme of the glycerol metabolic pathway (29), and subsequently the genes encoding the glycerol transporter (glpF), glycerol kinase (glpK), and the regulatory gene (glpR) (23) (Fig.…”

“…The same analyses indicated no probable ORFs on the opposite strand in this region. Second, a protein of the expected size is expressed in an Escherichia coli T7 expression system containing the sequences encompassing nt The map and genomic organization of the glpD region of the P. aeruginosa chromosome was derived from our previously published work (29) as well as from unpublished data (23). Abbreviations: D, glpD (glycerol-3-phosphate dehydrogenase); F, glpF (glycerol facilitator); K, glpK (glycerol kinase); M, glpM (this study); R, glpR (glp regulatory gene).…”

“…As a first step in elucidating the molecular organization and mode(s) of regulation of the glp genes of P. aeruginosa, we have recently cloned the glpD gene, encoding sn-glycerol-3-phosphate dehydrogenase, a key enzyme of the glycerol metabolic pathway (29), and subsequently the genes encoding the glycerol transporter (glpF), glycerol kinase (glpK), and the regulatory gene (glpR) (23) (Fig. 1A).…”

“…The glpD mutant strain PAO151 was obtained by insertion of the tetracycline resistance (Tc r )-encoding ⍀ element from pHP45⍀Tc (5) into the single NotI site (Fig. 1A) within glpD on pEB22⌬E1 (29). Similarly, strain PAO206 containing a chromosomal mutation of the glpD downstream region was obtained by insertion of a gentamicin resistance (Gm r )-encoding ⍀ element (27) at a NaeI site (marked in Fig.…”