The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages

phi CTX is a cytotoxin-converting phage isolated from Pseudomonas aeruginosa. In this study, we determined the complete nucleotide sequence of the phi CTX phage genome. The precise genome size was 35,538 bp with 21 base 5'-extruding cohesive ends. Forty-seven open reading frames (ORFs) were identified on the phi CTX genome, including two previously identified genes, ctx and int. Among them, 15 gene products were identified in the phage particle by protein microsequencing. The most striking feature of the phi CTX genome was an extensive homology with the coliphage P2 and P2-related phages; more than half of the ORFs (25 ORFs) had marked homology to P2 genes with 28.9-65.8% identity. The gene arrangement on the genome was also highly conserved for the two phages, although the G + C content and codon usage of most phi CTX genes were similar to those of the host P. aeruginosa chromosome. In addition, phi CTX was found to share several common features with P2, including the morphology, non-inducibility, use of lipopolysaccharide core oligosaccharide as receptor and Ca(2+)-dependent receptor binding. These findings indicate that phi CTX is a P2-like phage well adapted to P. aeruginosa, and provide clear evidence of the intergeneric spread and evolution of bacteriophages. Furthermore, comparative analysis of genome structures of phi CTX, P2 and other P2 relatives revealed the presence of several hot-spots where foreign DNAs, including the cytotoxin gene, were inserted. They appear to be deeply concerned in the acquisition of various genes that are horizontally transferred by bacteriophage infection.     

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.     

Molecular analysis of a cytotoxin-converting phage, φCTX, of Pseudomonas aeruginosa: structure of the attP–cos–ctx region and integration into the serine tRNA gene

The Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage φCTX. The genome of φCTX is a 35.5 kb double-stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of φCTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP–cos–ctx region. The termini of φCTX are 21-base 5′ extended-single-stranded DNAs. The ctxgene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP–cos–ctx region contains six kinds of repeats and integration host factor-binding sequences and showed sequence-directed static bends, suggesting its potential to form a highly ordered structure. In addition, φCTX was found to integrate into the serine tRNA gene which was mapped to the 43–45 min region on the P. aeruginosachromosome.     

Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12 with a randomly O-acetylated core region

The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and analyses by ESI FT-MS and NMR spectroscopy. The structures of the lipopolysaccharide core and the O-polysaccharide repeating unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established. The core was found to be randomly O-acetylated, most O-acetyl groups being located on the terminal rhamnose residue of the outer core region.     

The role of lipopolysaccharide as a receptor for some bacteriophages of Pseudomonas aeruginosa

Typing phages of the Colindale typing set for Pseudomonas aeruginosa have been tested for the use of lipopolysaccharide (LPS) as a receptor. Studies using the reference strains of the International Antigenic Typing Scheme for O-serotypes of P. aeruginosa supported earlier indications that none of the phages were O-specific. Studies of the adsorption of phages to LPS showed that typing phages 16, 44, F8, 68, 109, 352, and 1214 (as well as other phages 2 and H22) were LPS-specific, but were not consistently adsorbed by isolated LPS from all sensitive strains. Water-soluble fractions from LPS did not adsorb phages and did not inhibit their neutralization by whole LPS. No endoglycosidase activity against LPS was detected for any phage. The significance of these results for the roles of LPS in the adsorption process and phage sensitivity are discussed.     

Common antigen lipopolysaccharide from Pseudomonas aeruginosa AK1401 as a receptor for bacteriophage A7.

A-band, a D-rhamnose-containing common lipopolysaccharide antigen isolated from Pseudomonas aeruginosa AK1401, was found to be a receptor for bacteriophage A7. The phage-borne rhamnanase was capable of hydrolyzing the A-band to expose core-lipid A containing only two or three rhamnose repeats. Interaction of the hydrolyzed A-band with core- or lipid A-specific monoclonal antibodies revealed that common epitopes exist in the inner core and lipid A regions, while the outer core of A-band appears to be different from that of the serotype-specific (B-band) lipopolysaccharide.     

The Pseudomonas aeruginosa algC gene encodes phosphoglucomutase, required for the synthesis of a complete lipopolysaccharide core.

We have constructed strains of Pseudomonas aeruginosa with mutations in the algC gene, previously shown to encode the enzyme phosphomannomutase. The algC mutants of a serotype O5 strain (PAO1) and a serotype O3 strain (PAC1R) did not express lipopolysaccharide (LPS) O side chains or the A-band (common antigen) polysaccharide. The migration of LPS from the algC mutant strains in Tricine-sodium dodecyl sulfate-polyacrylamide gels was similar to that of LPS from a PAO1 LPS-rough mutant, strain AK1012, and from a PAC1R LPS-rough mutant, PAC605, each previously shown to be deficient in the incorporation of glucose onto the LPS core (K. F. Jarrell and A. M. Kropinski, J. Virol. 40:411-420, 1981, and P. S. N. Rowe and P. M. Meadow, Eur. J. Biochem. 132:329-337, 1983). We show that, as expected, the algC mutant strains had no detectable phosphomannomutase activity and that neither algC strain had detectable phosphoglucomutase (PGM) activity. To confirm that the PGM activity was encoded by the algC gene, we transferred the cloned, intact P. aeruginosa algC gene to a pgm mutant of Escherichia coli and observed complementation of the pgm phenotype. Our finding that the algC gene product has PGM activity and that strains with mutations in this gene produce a truncated LPS core suggests that the synthesis of glucose 1-phosphate is necessary in the biosynthesis of the P. aeruginosa LPS core. The data presented here thus demonstrate that the algC gene is required for the synthesis of a complete LPS core in two strains with different LPS core and O side chain structures.     

Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS: [structure: see text] where Kdo and Hep are 3-deoxy-D-manno-octulosonic acid and L-glycero-D-manno-heptose, respectively; all sugars are in the pyranose form and have the D configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, HepI being predominantly bisphosphorylated and HepII monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an L-alanyl group, HepII is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPS-rough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined.     

New temperate Pseudomonas aeruginosa phage, phi297: Specific features of genome structure

The genome structure and some specific features of temperate Pseudomonas aeruginosa phage phi297 are considered. Analysis of sequencing data and genome annotation suggest that the phi297 genome displays a mosaic structure, which has formed through combining gene blocks from bacteria of taxonomically remote groups and/or their phages. The results of a comparison of the phi297 DNA homology level and pattern with the genome sequences of the currently known related P. aeruginosa bacteriophages are interpreted from the perspective of assumed active migration of these phages between different bacterial species.     

Synthesis of Pseudomonas aeruginosa lipopolysaccharide core antigens containing 7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranosyl residues.

The monosaccharide allyl 7-O-carbamoyl-L-glycero-alpha-D-manno- heptopyranoside, the reducing disaccharide 7-O-carbamoyl-L-glycero-alpha-D- manno-heptopyranosyl-(1-->3)-L-glycero-D-manno-heptopyranose and the disaccharides allyl 7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranosyl-(1-->3)-L-glycero- beta- and alpha-D-manno-heptopyranoside were prepared in good yields. The 7-O-carbamoyl substituent was regioselectively introduced via NH3-NH4HCO3 treatment of a 6,7-O-carbonate group. Glycosylation steps were carried out using Me3SiOTf or BF3.Et2O promoted coupling of allyl alcohol with trichloroacetimidate or fluoride glycosyl donors, respectively. The deprotected allyl glycosides were reacted with cysteamine to afford spacer glycosides which were subsequently linked to bovine serum albumin. The artificial antigens which are related to the dephosphorylated heptose region of the lipopolysaccharide core region from Pseudomonas aeruginosa classified into RNA group I may be used for the characterization of monoclonal antibodies directed against inner core epitopes of human-pathogenic Pseudomonas species.     

Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide.

WaaP of P. aeruginosa is a crucial sugar kinase that phosphorylates HepI in the inner core region of lipopolysaccharide (LPS). WaaP shares homology with eukaryotic protein kinases in the conserved functional motifs (I-IX), indicating that it is also a protein kinase. This interpretation is substantiated by several lines of evidence including the following: (i) site-directed mutagenesis on catalytic domain residues abrogated the protein kinase activity; (ii) positive reaction in immunoblotting with anti-phosphotyrosine monoclonal antibody PY20; (iii) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and proteolytic peptide mapping showing excess mass equivalent to eight phosphate substituents on the tyrosine residues in WaaP; and (iv) WaaP is capable of catalyzing tyrosine self-phosphorylation as well as phosphorylating an exogenous synthetic co-polymer poly(Glu, Tyr). Thus, WaaP possesses dual kinase functions, and it utilizes a catalytic mechanism similar to that of the eukaryotic protein kinases. WaaP was localized to the cytoplasm, suggesting that phosphorylation of the LPS core occurred prior to translocation to the periplasm and attachment of O-antigen. A chemiluminescence-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the kinetics of the WaaP sugar kinase activity, and the results showed that the K(m) was 0.22 mm for ATP and 14.4 microm for hydrofluoric acid-treated LPS, V(max) was 408.24 pmol min(-1), and k(cat) was 27.23 min(-1).     

Monoclonal antibodies that distinguish inner core, outer core, and lipid A regions of Pseudomonas aeruginosa lipopolysaccharide.

In order to examine the immunochemistry of the core-lipid A region of Pseudomonas aeruginosa lipopolysaccharide (LPS), monoclonal antibodies (MAbs) specific for this region were produced in mice. Immunogen was prepared by coating a rough mutant of P. aeruginosa with column-purified core oligosaccharide fractions in order to enhance the immune response to the LPS core-lipid A region. Fourteen hybridoma clones were isolated, characterized, and further divided into three groups on the basis of their reactivities to rough LPS antigens in both enzyme-linked immunosorbent assays and Western immunoblots. In addition, another MAb, 18-19, designated group 1, was included in this study for defining core-lipid A epitopes. MAb 18-19 recognizes the LPS core-plus-one O-repeat unit of the serologically cross-reactive P. aeruginosa O2, O5, and O16. Group 2 MAbs are specific for the LPS outer core region and reacted with P. aeruginosa O2, O5, O7, O8, O10, O16, O18, O19, and O20, suggesting that these serotypes share a common outer core type. Group 3 MAbs recognize the inner core region and reacted with all 20 P. aeruginosa serotypes as well as with other Pseudomonas species, revealing the conserved nature of this region. Group 4 MAbs are specific for lipid A and reacted with all gram-negative organisms tested. Immunoassays using these MAbs and well-defined rough mutants, in addition to the recently determined P. aeruginosa core structures, have allowed us to precisely define immunodominant epitopes within the LPS core region.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Promises and pitfalls of Pseudomonas aeruginosa lipopolysaccharide as a vaccine antigen

Antibodies directed to the Pseudomonas aeruginosa lipopolysaccharide (LPS) O-antigens have clearly shown to mediate the most effective immunity to infection caused by LPS-smooth strains. Such strains are major causes of disease in immunocompromised hosts such as burn or cancer patients, individuals in intensive care units, and those who utilize extended-wear contact lenses. Yet producing an effective vaccine composed of non-toxic, immunogenic polysaccharides has been challenging. The chemical diversity among the different O-antigens representative of the 20 major serotypes, plus additional diversity among some O-antigens representing variant subtype antigens, translates into a large degree of serologic variability that increases the complexity of O-antigen specific vaccines. Further complications come from the poor immunogenicity of the major protective epitope expressed by some O-antigens, and a large degree of diversity in animal responses that preclude predicting the optimal vaccine formulation from such studies. Nonetheless human trials over the years of vaccines eliciting O-antigen immunity have been encouraging, though no vaccine has yet been fully evaluated and found to be clinically efficacious. Newer vaccine approaches such as using polysaccharide-protein conjugates and passive therapy with monoclonal or polyclonal immune sera offer some additional means to try and produce an effective immunotherapeutic reagent for this problematic pathogen.     

Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae

The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi. This phage transmits ctxAB genes between V. cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor. In investigating new forms of ctxAB transmission, we found that V. cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism. This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid). The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA. The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression. Infection and lysogenization with HybP phi revert the V. cholerae live attenuated vaccine strain 1333 to virulence. Our results reinforce that TCP is not indispensable for the acquisition of CTX phi. Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V. cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.     

Physical map of Pseudomonas aeruginosa phage phi kF77 genome. Localization of sites sensitive to restriction endonucleases

A physical map of the P. aeruginosa bacteriophage phi kF77 has been constructed using the restriction endonucleases SalI, HindIII, EcoRI, EcoRV, MuI, XbaI, ClaI. The phi kF77 DNA is resistant to cleavage by the restriction endonucleases BamHI, BglII, HpaI, PstI, PvuII, SmaI, XhoI.     

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.     

Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide.

The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy. Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O-polysaccharide repeating unit (B) or with a long-chain O-polysaccharide. Therefore, of two core glycoforms, only glycoform 2 accepts the O-polysaccharide. In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3-deoxy-d-manno-octulosonic acid, l-glycero-d-manno-heptose, 7-O-carbamoyl-l-glycero-d-manno-heptose, 2-acetamido-3-O-acetyl-2-deoxygalacturonamide, 2-formamido-2-deoxygalacturonamide, 2-acetamido-2,6-dideoxyglucose and 2-(l-alanylamino)-2-deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the LPS molecules, an O-acetyl group is present in the outer core region at unknown position. The site and the configuration of the linkage between the O-polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P. aeruginosa immunotype 1. The QuiNAc residue linked to the Rha residue of the core was found to have the beta configuration, whereas in the interior repeating units of the O-polysaccharide this residue is in the alpha-configuration. The data obtained are in accordance with the initiation of biosynthesis of the O-polysaccharide of P. aeruginosa O6, which is closely related to immunotype 1, by transfer of d-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide.