Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH.

Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37 degrees C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30 degrees C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30 degrees C, and it is fully defective in Ndk production.     

Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes.

Chromosomal DNA from group I Pseudomonas species, Azotobacter vinelandii, Azomonas macrocytogens, Xanthomonas campestris, Serpens flexibilis, and three enteric bacteria was screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes (algA, pmm, algD, and algR1). All the group I Pseudomonas species tested (including alginate producers and nonproducers) contained sequences homologous to all the P. aeruginosa alg genes used as probes, with the exception of P. stutzeri, which lacked algD. Azotobacter vinelandii also contained sequences homologous to all the alg gene probes tested, while Azomonas macrocytogenes DNA showed homology to all but algD. X. campestris contained sequences homologous to pmm and algR1 but not to algA or algD. The helical bacterium S. flexibilis showed homology to the algR1 gene, suggesting that an environmentally responsive regulatory gene similar to algR1 exists in S. flexibilis. Escherichia coli showed homology to the algD and algR1 genes, while Salmonella typhimurium and Klebsiella pneumoniae failed to show homology with any of the P. aeruginosa alg genes. Since all the organisms tested are superfamily B procaryotes, these results suggest that within superfamily B, the alginate genes are distributed throughout the Pseudomonas group I-Azotobacter-Azomonas lineage, while only some alg genes have been retained in the Pseudomonas group V (Xanthomonas) and enteric lineages.     

Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes.

Chromosomal DNA from group I Pseudomonas species, Azotobacter vinelandii, Azomonas macrocytogens, Xanthomonas campestris, Serpens flexibilis, and three enteric bacteria was screened for sequences homologous to four Pseudomonas aeruginosa alginate (alg) genes (algA, pmm, algD, and algR1). All the group I Pseudomonas species tested (including alginate producers and nonproducers) contained sequences homologous to all the P. aeruginosa alg genes used as probes, with the exception of P. stutzeri, which lacked algD. Azotobacter vinelandii also contained sequences homologous to all the alg gene probes tested, while Azomonas macrocytogenes DNA showed homology to all but algD. X. campestris contained sequences homologous to pmm and algR1 but not to algA or algD. The helical bacterium S. flexibilis showed homology to the algR1 gene, suggesting that an environmentally responsive regulatory gene similar to algR1 exists in S. flexibilis. Escherichia coli showed homology to the algD and algR1 genes, while Salmonella typhimurium and Klebsiella pneumoniae failed to show homology with any of the P. aeruginosa alg genes. Since all the organisms tested are superfamily B procaryotes, these results suggest that within superfamily B, the alginate genes are distributed throughout the Pseudomonas group I-Azotobacter-Azomonas lineage, while only some alg genes have been retained in the Pseudomonas group V (Xanthomonas) and enteric lineages.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.