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

Abstract

In a mucB (algN) genetic background, insertion of an ⍀ element ϳ200 bp downstream of glpD, encoding sn-glycerol-3-phosphate dehydrogenase from Pseudomonas aeruginosa, had an adverse effect on alginate biosynthesis from various carbon sources. The insertion inactivated glpM, a gene encoding a 12,040-M r hydrophobic protein containing 109 amino acids. This protein, which was expressed in a T7 RNA polymerase expression system, appears to be a cytoplasmic membrane protein.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 16 and 17). Whereas over the past few years significant progress has been made toward (i) understanding the complex regulation of the expression of the alginate biosynthetic genes and (ii) characterization of the gene products involved in the biosynthetic process (16,17), little is known about the origin(s) of the carbon moieties found in the alginate molecule. The desire to understand these origins is also driven by our lack of understanding of the availability of carbon in the cystic fibrosis lung, the environment in which mucoid derivatives of P. aeruginosa are most often found and in which they afflict the most damage. Of particular interest to our studies are the previously reported findings that triose phosphates are obligate intermediates in the biosynthesis of alginate (1) and that fructose 1,6-bisphosphate aldolase is essential for this to occur (2). We are therefore focussing our efforts on one peripheral carbon metabolic pathway which is capable of directly providing these triose phosphate intermediates (2), namely the glycerol metabolic pathway.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. 1A). In this communication, we report the observation that insertions in the glpD region have an adverse effect on alginate biosynthesis and that this may be due to inactivation of a closely linked gene, glpM.Insertions in the glpD region have an adverse effect on alginate biosynthesis. Construction of insertion mutants in the glpD region of wild-type strain PAO1 (8) was done by a previously described sacB-based gene replacement procedure (26). 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...

Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene

“…This argument was further supported by our recent discovery of glpM, which is located immediately downstream of glpD and whose product is required for efficient alginate biosynthesis from various carbon sources (29). Although glpM was suspected to be a part of the glp regulon, the nature of its regulation could not conclusively be demonstrated in the absence of a defined glpR mutation.…”

“…Sequence analysis of a 1-kb SphI subclone of a chromosomal 4.5-kb EcoRI fragment (Fig. 1A) (28) containing the sequences upstream of the previously characterized glpD (27) and glpM (29) genes revealed an open reading frame with significant homology to the E. coli glpR gene. The sequence of this SphI fragment and its downstream region is presented in Fig.…”

“…It is preceded by a good consensus Shine-Dalgarno sequence and a putative Ϫ10 and [27]); F and K, glpF and glpK (membrane-associated glycerol diffusion facilitator and glycerol kinase, respectively [28]); M, glpM (membrane protein affecting alginate synthesis [29]); R, glpR (glp regulatory gene [this study]); X and Y, orfX and orfY (a transcriptional regulator and carbohydrate kinase, respectively, with unknown functions [28]). (B) Nucleotide sequence of an SphI-EcoRI fragment containing glpR.…”

“…Workers in several laboratories, including that of Williams et al (41) and ours (23,27,29), have argued the probable importance of glycerol as a source of carbon for growth and alginate synthesis within the lung. This argument was further supported by our recent discovery of glpM, which is located immediately downstream of glpD and whose product is required for efficient alginate biosynthesis from various carbon sources (29).…”

Bacterial alginate biosynthesis – recent progress and future prospects

“…At least 24 genes have been directly implicated in alginate biosynthesis in P. aeruginosa (Table 1) and there is good evidence that others may also be involved, e.g. glpM (Schweizer et al, 1995). It is difficult to be precise about the number of genes that are solely associated with alginate biosynthesis as it is now clear, for example, that some of the control genes act globally and encode proteins such as alternative cr factors .…”