Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase

Abstract

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.

Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa

“…GMDH converts GDPmannose into GDPmannuronate, a direct precursor for alginate polymerization (14). The biosynthetic steps preceding GDPmannuronate are shared with lipopolysaccharide biosynthetic pathways and, although necessary for alginate synthesis, are not entirely alginate specific (10,26). In keeping with the pivotal role of GDPmannose dehydrogenase, transcriptional activation of algD is always associated with the mucoid status of P. aeruginosa and represents a committing step towards increased alginate synthesis.…”

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide

“…Mutants devoid of 0 antigen were considerably more sensitive to serum components and phagocytic effects than their parent strains were. It has been recently demonstrated that the enzyme PMM is involved in LPS synthesis in P. aeruginosa PAO1 (13). Studies with LPS-rough mutant strain AK1012 showed a lack of glucose and rhamnose residues in the LPS and that the probable block in LPS synthesis was in the attachment of glucose or rhamnose residues to the galactosamine in the LPS core (15).…”

Structural basis for alginate secretion across the bacterial outer membrane

“…Of the thirteen genes required for the biosynthesis and secretion of alginate by P. aeruginosa, twelve are located on the tightly regulated algD operon. The algC gene, located elsewhere in the genome, has additional roles in lipopolysaccharide (LPS) and rhamnolipid biosynthesis (7,8). Biosynthesis of alginate starts with the production of the activated sugar-nucleotide precursor GDP-mannuronic acid from fructose-6-phosphate via the concerted actions of the AlgA, AlgC, and AlgD enzymes (reviewed in ref.…”