The effects of chlorhexidine, povidone-iodine and vancomycin on growth and biofilms of pathogens that cause prosthetic joint infections: an in-vitro model.

BACKGROUND: Chlorhexidine gluconate (CHG) and povidone-iodine (PI) are commonly used to prevent prosthetic joint infection (PJI) during total joint replacement; however, their effective concentrations and impact on biofilms are not well defined. AIM: To determine: (1) the in-vitro minimum inhibitory concentration of CHG and PI against model PJI-causing organisms and clinical isolates; (2) their impact on biofilm formation; (3) whether there is a synergistic benefit to combining the two solutions; and (4) whether adding the antibiotic vancomycin impacts antiseptic activity. METHODS: We measured in-vitro growth and biofilm formation of Staphylococcus epidermidis, meticillin-sensitive and meticillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans, as well as recent clinical isolates, in the presence of increasing concentrations of CHG and/or PI. Checkerboard assays were used to measure potential synergy of the solutions together and with vancomycin. FINDINGS: CHG and PI inhibited growth and biofilm formation of all model organisms tested at concentrations of 0.0004% and 0.33% or lower, respectively; highly dilute concentrations paradoxically increased biofilm formation. The solutions did not synergize with one another and acted independently of vancomycin. CONCLUSION: CHG and PI are effective at lower concentrations than typically used, establishing baselines to support further clinical trials aimed at optimizing wound disinfection. There is no synergistic advantage to using both in combination. Vancomycin is effective at inhibiting the growth of S. epidermidis and S. aureus; however, it stimulates P. aeruginosa biofilm production, suggesting in the rare case of P. aeruginosa PJI, it could exacerbate infection.

Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.

Type IVa pili are bacterial nanomachines required for colonization of surfaces, but little is known about the organization of proteins in this system. The Pseudomonas aeruginosa pilMNOPQ operon encodes five key members of the transenvelope complex facilitating pilus function. While PilQ forms the outer membrane secretin pore, the functions of the inner membrane-associated proteins PilM/N/O/P are less well defined. Structural characterization of a stable C-terminal fragment of PilP (PilP(Δ71)) by NMR revealed a modified ß-sandwich fold, similar to that of Neisseria meningitidis PilP, although complementation experiments showed that the two proteins are not interchangeable likely due to divergent surface properties. PilP is an inner membrane putative lipoprotein, but mutagenesis of the putative lipobox had no effect on the localization and function of PilP. A larger fragment, PilP(Δ18-6His), co-purified with a PilN(Δ44)/PilO(Δ51) heterodimer as a stable complex that eluted from a size exclusion chromatography column as a single peak with a molecular weight equivalent to two heterotrimers with 1:1:1 stoichiometry. Although PilO forms both homodimers and PilN-PilO heterodimers, PilP(Δ18-6His) did not interact stably with PilO(Δ51) alone. Together these data demonstrate that PilN/PilO/PilP interact directly to form a stable heterotrimeric complex, explaining the dispensability of PilP's lipid anchor for localization and function.

Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa.

Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.

Conserved, unstructured regions in Pseudomonas aeruginosa PilO are important for type IVa pilus function.

Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilOΔ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous ß-strand in the second αßß motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.

Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals.

Escherichia coli may become resistant to cephamycines and oxyimino cephalosporins by virtue of promotor and attenuator mutations or because they have acquired mobilized beta-lactamases from other gram-negative bacilli. This study examined Canadian strains to determine how often promotor and/or attenuator mutations account for this mechanism of resistance and the extent to which clonal spread of these organisms has occurred. We sequenced the promotor and attenuator region of 30 strains resistant to cefoxitin. Twenty-two strains had promotor mutations, 26 had attenuator mutations. Most promotor mutations resulted either in a change in the -35 promotor region towards the E. coli sigma 70 consensus sequence or in the creation of a new consensus hexamer upstream. Eight strains had mutations that increased the typical ampC 16-nucleotide spacer region to the consensus 17- or an 18-nucleotide sequence. Of the attenuator mutations, most did not substantially affect the attenuator loop. Several of the mutations have previously been described in South Africa, Scandinavia, and France. There was evidence that strains bearing certain mutations were clonally disseminated; however, the 11 strains bearing a complex set of attenuator mutations were not. The majority of cephamycin resistant E. coli strains in Toronto have attenuator and/or promotor mutations upstream of the chromosomal ampC gene.

Pseudomonas aeruginosa B-band lipopolysaccharide genes wbpA and wbpI and their Escherichia coli homologues wecC and wecB are not functionally interchangeable.

The O antigen unit of Pseudomonas aeruginosa serotype O5 is a complex trisaccharide containing 2-acetamido-3-acetiminido-2, 3-dideoxy-beta-D-mannuronic acid, 2-acetimido-3-acetimido-2, 3-dideoxy-beta-D-mannuronic acid, and 2-acetimido-2, 6-deoxy-beta-D-galactosamine. Specific knockout mutations in the putative UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) epimerase gene, wbpI, or the putative UDP-D-N-acetylmannosamine dehydrogenase gene, wbpA, resulted in strains that no longer produced B-band lipopolysaccharide, confirming the essential roles of these genes in B-band O antigen synthesis. Despite approximately 50% similarity of wbpI and wbpA to the Escherichia coli genes wecB (rffE) and wecC (rffD) involved in enterobacterial common antigen synthesis, cross-complementation experiments were not successful. These results imply that the P. aeruginosa UDP-D-GlcNAc precursor may be di-N-acetylated prior to further modification, preventing the E. coli enzymes from recognizing it as a substrate.

Genetic and biochemical characterization of an operon involved in the biosynthesis of 3-deoxy-D-manno-octulosonic acid in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa serotype O5 (PAO1) genomic DNA fragment that was able to complement a temperature-sensitive mutation in the 3-deoxy-D-manno-octulosonic acid (Kdo) 8-P synthase gene (kdsA) of Salmonella enterica serovar typhimurium was cloned. Nucleotide sequence analysis revealed the presence of a potential operon with the gene order pyrG, kdsA, eno. PyrG catalyzes the synthesis of the nucleotide cytidine triphosphate, while Eno catalyzes the formation of phosphoenolpyruvate from phosphoglycerate during glycolysis. Phosphoenolpyruvate is one of the substrates for Kdo-8-P biosynthesis by KdsA and cytidine triphosphate is the nucleotide used to activate Kdo prior to its transfer to lipid A. pyrG and eno are important for many metabolic pathways and it is interesting to find them linked to kdsA. A sigma 70-like promoter was found upstream of pyrG and evidence was provided to show that this promoter was responsible for the initiation of transcription of the genes in this operon. These genes mapped to 28.2-29.9 min on the 75-min PAO1 chromosome, unlinked to other lipopolysaccharide biosynthetic gene clusters.

PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin.

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.

Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.

Molecular characterization of an RTX toxin determinant from Actinobacillus suis.

RTX cytolysins are a family of calcium-dependent, pore-forming, secreted toxins found in a variety of gram-negative bacteria. The prototypical member of this family is the alpha-hemolysin of Escherichia coli. The RTX genetic determinants from seven members of the family Pasteurellaceae, Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and A. pleuropneumoniae serotypes 1,5,7, and 9 were previously cloned and sequenced. Using the leukotoxin determinant from P. haemolytica serotype A1 as a probe, we detected the presence of RTX-type determinants in Actinobacillus suis, A. equuli, and A. lignieresii of the family Pasteurellaceae. All three species elaborate proteins of approximately 104 to 110 kDa that are recognized by polyclonal antisera against the 104-kDa hemolysin of A. pleuropneumoniae serotype 1. An RTX determinant of A. suis isolate 3714 was cloned and sequenced and was found to be almost identical to the RTX determinant of A. pleuropneumoniae serotypes 5 and 9. In addition, the determinant is not composed of four contiguous genes, as had been reported for most other RTX determinants; instead, the genes encoding the two proteins responsible for secretion of the toxin are at a locus distinct from that containing the toxin structural and activation genes.

Issues surrounding the prevention and management of device-related infections.

The use of biomaterial devices in all aspects of modern medicine has increased exponentially in the past three decades. Device-related infections constitute one of the main impediments to their long-term use. We discuss the pathogenesis, prevention, and management of catheter-associated urinary tract infection (CAUTI). The recent innovations in biomaterial design and surface coatings intended to prevent colonization of the device surface are presented. Despite these significant advances, the ability to protect these surfaces beyond the short term presents a continued challenge to investigators.

Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5.

The wbp cluster of Pseudomonas aeruginosa O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in the wbp cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or "flippase." Insertion of a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal wzx gene replacement mutations. Surprisingly, inactivation of wzx also led to a marked delay in production of the ATP-binding cassette-transporter-dependent, D-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103-1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of wzx mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

Molecular analysis of the leukotoxin determinants from Pasteurella haemolytica serotypes 1 to 16.

All sixteen serotypes of Pasteurella haemolytica were shown to produce a leukotoxin protein which is immunologically related to the well-characterized serotype 1 leukotoxin. All of the leukotoxins were weakly hemolytic and were able to bind to BL-3 target cells. The leukotoxin determinants were characterized by Southern blot hybridization by use of the previously cloned serotype 1 determinant as the probe, and a number of distinct classes were identified. The leukotoxin determinants from serotypes 2, 3, and 11 were cloned. Nucleotide sequence analysis of the lktC and lktA genes of the serotype 3 and 11 determinants revealed nucleotide substitutions throughout the coding sequences. A comparison of the lktC and lktA genes and deduced proteins of serotypes 1, 3, and 11 showed that they are highly homologous.

Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.

Functional conservation of the polysaccharide biosynthetic protein WbpM and its homologues in Pseudomonas aeruginosa and other medically significant bacteria.

WbpM is a highly conserved protein involved in synthesis of the O antigens of Pseudomonas aeruginosa. Homologues of this protein have been identified in a large number of bacteria, and they can be divided into two subfamilies: subfamily 1, including WbpM, contains large proteins ( approximately 600 amino acids), while subfamily 2, typified by HP0840 (FlaA1) of Helicobacter pylori, contains smaller proteins ( approximately 350 amino acids) homologous to the C termini of proteins in subfamily 1. Analysis of knockout mutants of wbpM in P. aeruginosa serotypes O3, O10, O15, and O17 showed that although all 20 serotypes of P. aeruginosa possess wbpM, it is not universally required for O-antigen biosynthesis. Homologous genes from Bordetella pertussis (wlbL), Staphylococcus aureus (cap8D), and H. pylori (flaA1) complemented a P. aeruginosa O5 wbpM mutant to various degrees. These conserved proteins may represent interesting targets for the design of inhibitors of bacterial exopolysaccharide biosynthesis.

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster.

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2-5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA-wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequences, IS 1209, is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.

Low-virulence Citrobacter species encode resistance to multiple antimicrobials.

Citrobacter spp. are gram-negative commensal bacteria that infrequently cause serious nosocomial infections in compromised hosts. They are often resistant to cephalosporins due to overexpression of their chromosomal beta-lactamase. During a recent study of multidrug-resistant Enterobacteriaceae (MDRE) in solid-organ transplant patients, we found that almost half of patients colonized with MDRE carried one or more cefpodoxime-resistant Citrobacter freundii, Citrobacter braakii, or Citrobacter amalonaticus strains. Pulsed-field gel electrophoresis showed that 36 unique strains of Citrobacter were present among 32 patients. Genetic and phenotypic analysis of the resistance mechanisms of these bacteria showed that the extended-spectrum beta-lactamase (ESBL) SHV-5 or SHV-12 was encoded by 8 strains (26%) and expressed by 7 strains (19%). A number of strains were resistant to other drug classes, including aminoglycosides (28%), trimethoprim-sulfamethoxazole (31%), and fluoroquinolones (8%). PCR and DNA analysis of these multiresistant strains revealed the presence of class I integrons, including the first integrons reported for C. braakii and C. amalonaticus. The integrons encoded aminoglycoside resistance, trimethoprim resistance, or both. Despite the prevalence of MDR Citrobacter spp. in our solid-organ transplant patients, only a single infection with a colonizing strain was recorded over 18 months. Low-virulence Citrobacter spp., which can persist in the host for long periods, could influence pathogen evolution by accumulation of genes encoding resistance to multiple antimicrobial classes.

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis.

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of alpha1-->2, alpha1-->3, alpha1-->3 linked D-rhamnose (Rha). The A-band polysaccharide is assembled by the alpha-D-rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two alpha1-->3 linked Rha residues and one alpha1-->2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3' end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3' end of the cluster and are predicted to encode a coenzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D-galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.

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

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O-antigen from O5 to O16 and O-acetylating the N-acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver-stained SDS-PAGE gels and displayed reactivity to an antibody specific for O-acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an alpha-polymerase inhibitor (iap); an O-acetylase (oac); and a beta-polymerase (wzybeta). The alpha-polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype-specific O5 B-band LPS and allows the phage-encoded beta-polymerase (Wzybeta) to form new beta-linked B-band LPS. The D3 phage also alters the LPS by the addition of O-acetyl groups to the FucNAc residue in the O-antigen repeat unit by the action of the D3 O-acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.