Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.
We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.
Goldberg
J B
JB
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115-5899.
Hatano
K
K
Pier
G B
GB
D015139:Blotting, Southern; D003227:Conjugation, Genetic; D016521:Electrophoresis, Gel, Pulsed-Field; D005816:Genetic Complementation Test; D015151:Immunoblotting; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases); D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D015183:Restriction Mapping
D008070:Lipopolysaccharides; D007535:Isomerases; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase