O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3

Abstract

The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.

Structural elucidation of the lipopolysaccharide core regions of the wild‐type strain PAO1 and O‐chain‐deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

“…It was shown by NMR analysis that for LPS from strain AK1401, the voidvolume fraction was comprised of D-rhamnan, derived from P. aeruginosa A-band common antigen. For the wild-type LPS from strain PAO1, the void-volume fraction comprised of a mixture of the O-antigen [32] and D-rhamnan.…”

Prevalence and distribution of bacteriophage φAa DNA in strains of Actinobacillus actinomycetemcomitans

“…h + = hybridization of phage dpAa DNA probe with DNA from A. actinomycetemcomitans strain. -= no hybridization of phage &Aa DNA probe with DNA from A. actinomycetemcomitans strain.phages to code for virulence factors such as toxins, capsules, resistance to serum bacteriolysis and altered antigenicity[7][8][9][10][11][12][13][14][15][16], we were interested in the possibility that A. actinomycetemcomitans phages, particularly phage ~bAa, play a role in the pathogenicity of A. actinomycetemcomitans. In this regard, we wanted to examine the prevalence and distribution of phage c~Aa infection in strains of A. actinomycetemcomitans.…”

Three-component-mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3

“…In an attempt to identify the region of the D3 genome that contains the gene(s) responsible for seroconversion, a 12 kb fragment of the genome lacking phage structural and assembly genes was isolated. This region also contained a large open reading frame (ORF) with homology to O-acetyltransferases (OATs), which made it an attractive region to analyse, as the D3 lysogen (AK1380) produces O antigen containing O-acetylated FucNAc residues (Kuzio and Kropinski, 1983). The 12 kb fragment of D3 DNA was cloned into pUCP26 and transformed into P. aeruginosa PAO1.…”

“…P. aeruginosa O-antigen repeat unit structure for the O5 serogroup (serotypes O2, O5, O16, O18 and O20) and AK1380 (D3 lysogen). The structures are highly similar, differing only in the type of glycosidic linkage (a-Dversus b-D-N-acetylfucosamine), isomer (b-D-mannuronic versus a-L-guluronic) or the presence of O-acetyl groups at C-4 of the fucosamine moiety (adapted from Kuzio and Kropinski, 1983;Knirel and Kochetkov, 1994). G, guluronic acid; M, mannuronic acid; F, fucosamine; Ac, acetyl group.…”

“…For example, lysogenization of Salmonella anatum by bacteriophage 1 15 changes the glycosidic linkages of the O antigen from alpha to beta and prevents O-acetyl residues from being incorporated into the O-antigen polymer (Robbins et al, 1965;Losick, 1969). Our laboratory is interested in the lambdoid bacteriophage D3, which lysogenizes P. aeruginosa strain PAO1, causing two major alterations to the O antigen: the C4 position of the FucNAc residue is O-acetylated; and the linkage between O units is changed from a1-4 to b1-4 (Kuzio and Kropinski, 1983) (Fig. 1).…”