Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD)

Holmberg, Christina; Beijer, Lena; Rutberg, Blanka; Rutberg, Lars

doi:10.1099/00221287-136-12-2367

Cited by 90 publications

(55 citation statements)

References 46 publications

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“…The original clone (>30 kb) was found to be unstable in E. coli. This situation is reminiscent of the previously reported finding that the B. subtilis glpPKD region could not be recovered in nonmutated form from E. coli (11). However, a single clone containing ca.. 2.8 kb of contiguous P. aeruginosa chromosomal DNA could be recovered, and Southern analysis proved the presence of the analyzed region in the PAO1 chromosome.…”

Section: Cgcccatttcaagcagcaactcgcggccgccgacatcctgcacagcttcqqcqcqgtgcsupporting

confidence: 45%

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

Schweizer

1994

J Bacteriol

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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...

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Section: Cgcccatttcaagcagcaactcgcggccgccgacatcctgcacagcttcqqcqcqgtgcsupporting

confidence: 45%

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

Schweizer

1994

J Bacteriol

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“…3). The optimum pH of GK (7·5) is different from pH 9·0 observed in B. subtilis (Holmberg et al 1990) and Ps. aeruginosa (Heath and Gaudy 1978 ;McCowen et al 1981).…”

Section: Influence Of Ph and Temperaturementioning

confidence: 98%

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

Pasteris

Saad

1998

Lett Appl Microbiol

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S A AD . 1998. The pathway involved in glycerol dissimilation in Pediococcus pentosaceus N5p, a strain isolated from wine, includes glycerol kinase and NAD-independent glycerol-3P dehydrogenase. The properties of these enzymes were studied. Glycerol kinase activity was maximal at 28°C and pH 7·5 in 50 mmol l −1 TrisHCl buffer. The end-products of the reaction acted as competitive inhibitors while fructose-1,6-diphosphate was a non-competitive inhibitor. Mg 2+ was required for optimal enzyme activity. The Km values for both substrates were 0·11 and 0·37 mmol l −1 for glycerol and ATP, respectively. NAD-independent glycerol-3P dehydrogenase activity was maximal at 37°C and pH 7·5 in 100 mmol l −1 Tris-HCl buffer. The enzymatic activity was activated by KCN and bivalent cations as Mg 2+ and Ca 2+, but it was strongly inhibited by others. Dihydroxyacetone phosphate acted as competitive inhibitor while ATP and phosphenolpyruvate were non-competitive inhibitors.

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“…We have previously reported the cloning of a 7 kb EcoRI-PstI B. subtilis chromosomal DNA fragment which carries wild type alleles of gZpP, glpK and gZpD mutations, and the nucleotide sequences of gZpK and gZpD have been presented (Holmberg et al, 1990). The plasmid carrying the 7 kb fragment is a derivative of plasmid pHP13 and is called pLUM30.…”

Section: Introductionmentioning

confidence: 99%

The glpP and glpF genes of the glycerol regulon in Bacillus subtilis

Beijer

Nilsson

Holmberg

et al. 1993

Journal of General Microbiology

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The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P). The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined. glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP. A aA-type promoter and the transcriptional start point for glpFK were identified. In the 5' untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found. The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase. Expression of glpFK and glpD requires the inducer G3P and the glpP gene product. A 2.9 kb B. subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7. pLUM7 contains a functional glpP gene as shown by its ability to complement various gZpP mutants. Immediately upstream of glpP an open reading ffame is found (Owl). Disrupting ORFl by plasmid integration in the B. subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source. With the present report all B. subtilis glp genes located at 75" on the chromosomal map have been identified.

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Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD)

Cited by 90 publications

References 46 publications

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

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

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

The glpP and glpF genes of the glycerol regulon in Bacillus subtilis

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