title: Evidence that the algI/algJ gene cassette, required for O acetylation of Pseudomonas aeruginosa alginate, evolved by lateral gene transfer.
abstract: Pseudomonas aeruginosa strains, isolated from chronically infected patients with cystic fibrosis, produce the O-acetylated extracellular polysaccharide, alginate, giving these strains a mucoid phenotype. O acetylation of alginate plays an important role in the ability of mucoid P. aeruginosa to form biofilms and to resist complement-mediated phagocytosis. The O-acetylation process is complex, requiring a protein with seven transmembrane domains (AlgI), a type II membrane protein (AlgJ), and a periplasmic protein (AlgF). The cellular localization of these proteins suggests a model wherein alginate is modified at the polymer level after the transport of O-acetyl groups to the periplasm. Here, we demonstrate that this mechanism for polysaccharide esterification may be common among bacteria, since AlgI homologs linked to type II membrane proteins are found in a variety of gram-positive and gram-negative bacteria. In some cases, genes for these homologs have been incorporated into polysaccharide biosynthetic operons other than for alginate biosynthesis. The phylogenies of AlgI do not correlate with the phylogeny of the host bacteria, based on 16S rRNA analysis. The algI homologs and the gene for their adjacent type II membrane protein present a mosaic pattern of gene arrangement, suggesting that individual components of the multigene cassette, as well as the entire cassette, evolved by lateral gene transfer. AlgJ and the other type II membrane proteins, although more diverged than AlgI, contain conserved motifs, including a motif surrounding a highly conserved histidine residue, which is required for alginate O-acetylation activity by AlgJ. The AlgI homologs also contain an ordered series of motifs that included conserved amino acid residues in the cytoplasmic domain CD-4; the transmembrane domains TM-C, TM-D, and TM-E; and the periplasmic domain PD-3. Site-directed mutagenesis studies were used to identify amino acids important for alginate O-acetylation activity, including those likely required for (i) the interaction of AlgI with the O-acetyl precursor in the cytoplasm, (ii) the export of the O-acetyl group across the cytoplasmic membrane, and (iii) the transfer of the O-acetyl group to a periplasmic protein or to alginate. These results indicate that AlgI belongs to a family of membrane proteins required for modification of polysaccharides and that a mechanism requiring an AlgI homolog and a type II membrane protein has evolved by lateral gene transfer for the esterification of many bacterial extracellular polysaccharides.
authors.affiliation: Department of Microbiology, 109 Lewis Hall, Montana State University, Bozeman, MT 59717, USA. umbfm@montana.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Franklin
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Douthit
authors.forename: Marcella A
authors.initials: MA
authors.lastname: McClure
mesh_terms: D000107:Acetylation; D000464:Alginates / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D017124:Conserved Sequence; D019143:Evolution, Molecular; D022761:Gene Transfer, Horizontal; D020723:Glucuronic Acid / Q000378:metabolism*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D016415:Sequence Alignment; D017385:Sequence Homology
chemical_list: C098843:AlgI protein, Pseudomonas aeruginosa; C098844:AlgJ protein, Pseudomonas aeruginosa; D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D008565:Membrane Proteins; D020723:Glucuronic Acid