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.

FIG. 1.

Position of algI or dltB homologs within operons or putative operons. The blue arrows represent the algI or dltB homolog. All colored arrows indicate genes either demonstrated or likely to be on the same operon as algI or dltB. The green arrows show the genes for the type II membrane proteins with homology to AlgJ. The red arrows indicate genes for type II membrane proteins with conserved amino acid motifs to the N. meningitidis NMA1479 protein. The yellow arrows indicate genes with homology to DltD of Bacillus subtilis. Shown in purple are genes for type II membrane homologs of Bacillus anthracis and Magnetococcus sp. Also shown are the algF homologs (light blue), showing differing gene order for the alginate biosynthetic operon of P. aeruginosa and the putative cellulose biosynthetic operon of P. syringae. Shown in pink is dltA; the inverted gene order for Bacillus subtilis compared to Bordetella pertussis is also shown. The G+C contents of the genomic DNA was obtained from the Codon Usage Database (http://www.kazusa.or.jp/codon/) and compared to the G+C content of the algI homologs.

FIG. 2.

Sequence alignments of conserved motifs for the three subsets of type II membrane proteins that are genetically linked to algI or dltB. (A) AlgJ group; (B) NMA1479 group; (C) DltD group. The white letters highlighted in black show the conserved motifs surrounding an aspartate and an histidine residue, which are found in all three groups. The gray highlighted letters show conserved amino acids within each group. Asterisks indicate amino acids of AlgJ from P. aeruginosa that reduce alginate O acetylation at least threefold when mutated. The circle indicates an amino acid that when mutated does not affect alginate O acetylation.

FIG. 3.

Phylogenetic analysis of AlgI and DltB homologs (shown on the left-hand side) and of the type II membrane proteins encoded by genes adjacent to algI or dltB (right-hand side). Trees were constructed by using neighbor-joining and bootstrapping analysis of aligned sequences. Filled circles indicate branch points with bootstrap support of >90%. Open circles indicate branch points with bootstrap values of >70%. Branch points without circles had bootstrap values of 50 to 75%. The yellow shaded region shows the phylogeny of the DltB proteins and their genetically linked DltD protein. The green-shaded region shows the phylogeny of a subset of AlgI proteins (left) and their linked AlgJ homologs (right). The red-shaded region shows a subset of AlgI homologs and their linked homologs to NMA1479 of N. meningitidis. The pink-shaded region shows one of the Bacillus anthracis AlgI and the Magnetococcus sp. AlgI homologs that are linked to genes for type II membrane proteins related to each other but not related to the type II membrane proteins of the other three groups. Desulfitobacterium hafniense encodes an AlgI homolog closely related to the P. aeruginosa AlgI clade but a type II membrane protein related to N. meningitidis NMA1479. Also shown are AlgI/DltB homolog eukaryotes. Classifications: C, Clostridiales; B, Bacillales; L, Lactobacillales; β, β-proteobacteria; γ, γ-proteobacteria; δ, δ-proteobacteria; α, α-proteobacteria; M, Magnetococcus sp.; GSB, green sulfur bacteria; S, Spirochaetales; E, eukaryotes.

FIG. 4.

FTIR spectra of alginate purified from mucoid P. aeruginosa strains showing presence or absence of ester linkages to O-acetyl groups. Alginates from wild-type strain FRD1 (A), FRD1176 algJ6Δ with control plasmid pMF54 (B), and FRD1176 (algJ6Δ) (C to I) with plasmids containing point mutations as indicated. The molar ratios of O-acetyl groups to uronic acid residues were determined by the colorimetric methods described in Materials and Methods. The data represent the averages for three independent strains containing the designated plasmid.

FIG. 5.

Sequence alignment of the TM domains TM-C, TM-D, and TM-E. Black letters within the sequence alignment indicate the TM regions predicted by TMHMM program (37). The white letters shaded in black are amino acids conserved throughout the AlgI/DltB homologs. The white letters shaded in gray are amino acids conserved throughout the DltB homologs. Asterisks indicate amino acids subjected to site-directed mutagenesis, where the mutation resulted in at least a fourfold decrease in alginate O acetylation. The circles indicate an amino acid change that had little effect on alginate O acetylation.

FIG. 6.

Sequence alignment showing ordered motifs in the cytoplasmic domain 4 (CP-4). The symbols are similar to those in Fig. 5, with black shaded areas showing amino acids with identity through the AlgI homologs and extending into the DltB homologs, and the gray highlights showing amino acids with identity through the DltB homologs and extending into the AlgI homologs. Asterisks indicate amino acids subjected to site-directed mutagenesis, where the mutation resulted in at least a fourfold decrease in alginate O acetylation. Circles indicate amino acid change that had little effect on alginate O acetylation.

FIG. 7.

Model of AlgI from P. aeruginosa, showing the highly conserved ordered series of motifs (shaded gray). Filled circles indicate the sites where amino acid substitutions resulted in at least a fourfold decrease in alginate O-acetylation activity. Open circles indicate conserved amino acid that when mutated do not affect alginate O acetylation.