O-antigen Chain Research Articles

Overproduction of DNA Adenine Methyltransferase Alters Motility, Invasion, and the Lipopolysaccharide O-Antigen Composition of Yersinia enterocolitica

DNA adenine methyltransferase (Dam) not only regulates basic cellular functions but also interferes with the proper expression of virulence factors in various pathogens. We showed previously that for the human pathogen Yersinia enterocolitica, overproduction of Dam results in increased invasion of epithelial cells. Since invasion and motility are coordinately regulated in Y. enterocolitica, we analyzed the motility of a Dam-overproducing (Dam(OP)) strain and found it to be highly motile. In Dam(OP) strains, the operon encoding the master regulator of flagellum biosynthesis, flhDC, is upregulated. We show that the increased invasion is not due to enhanced expression of known and putative Y. enterocolitica invasion and adhesion factors, such as Inv, YadA, Ail, Myf fibrils, Pil, or Flp pili. However, overproduction of Dam no longer results in increased invasion for an inv mutant strain, indicating that Inv is necessary for increased invasion after overproduction of Dam. Since we show that overproduction of Dam results in an increased amount of rough lipopolysaccharide (LPS) molecules lacking O-antigen side chains, this implies that reduced steric hindrance by LPS might contribute to increased invasion by a Y. enterocolitica Dam(OP) strain. Our data add an important new aspect to the various virulence-associated phenotypes influenced by DNA methylation in Y. enterocolitica and indicate that Dam targets regulatory processes modulating the composition and function of the bacterial surface.

Porphyromonas gingivalis galEIs Involved in Lipopolysaccharide O-Antigen Synthesis and Biofilm Formation

Porphyromonas gingivalis is a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalis mutants were constructed by insertion of an ermF-ermAM cassette; among these mutants, the galE mutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galE mutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galE mutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galE mutant had an intensity 4.5-fold greater than those of the wild type. Further, the galE mutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galE mutation led to a partial recovery of the parental phenotypes. We concluded that the galE gene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalis and considered that our findings of a relationship between the function of the P. gingivalis galE gene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.

The structural basis for the serospecificity ofActinobacillus suisserogroup O:2

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1-->6)-beta-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: -->3)-beta-D-Galp-(1-->4)-[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc-(1-->. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.

Overexpression and characterization of Wzz of Escherichia coli O86:H2

O-Antigen plays a critical role in the bacterium-host interplay, the chain length is an important factor in O-antigen functions. Wzz protein is responsible for O-antigen chain length regulation, but the mechanism is still unknown. Here, we overexpressed the Wzz of Escherichia coli O86:H2 in wzz mutant O86:H2 strain, the yield can achieve 15 mg/L. The recombinant Wzz was purified to 99% purity in dodecylmaltoside by sequential Ni-affinity chromatography and anion-exchange. Size exclusion chromatography and in vivo cross-linking experiments both showed that Wzz formed tetramer. Furthermore, analysis with circular dichroism revealed that the predominant structural composition in Wzz is α-helices, and incubation with O-antigen significantly changed Wzz conformation. The results suggested that Wzz protein can interact with O-antigen.

Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants

SUMMARY Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of potato. It can suppress soil-borne plant diseases by siderophore-mediated competition for iron, but it has also been reported to result in induced systemic resistance (ISR) in Arabidopsis thaliana. Bacterial determinants of this strain involved in inducing systemic resistance in Arabidopsis were investigated using a Tn5 transposon mutant defective in biosynthesis of the fluorescent siderophore pseudobactin, a non-motile Tn5 mutant lacking flagella, and a spontaneous phage-resistant mutant lacking the O-antigenic side chain of the lipopolysaccharides (LPS). When using Pseudomonas syringae pv. tomato as the challenging pathogen, purified pseudobactin, flagella and LPS all triggered ISR. However, the mutants were all as effective as the parental strain, suggesting redundancy in ISR-triggering traits in WCS358. The Botrytis cinerea-tomato, B. cinerea-bean and Colletotrichum lindemuthianum-bean model systems were used to test further the potential of P. putida WCS358 to induce ISR. Strain WCS358 significantly reduced disease development in all three systems, indicating that also on tomato and bean WCS358 can trigger ISR. In both tomato and bean, the LPS mutant had lost the ability to induce resistance, whereas the flagella mutant was still effective. In bean, the pseudobactin mutant was still effective, whereas this mutant has lost its effectivity in tomato. In both bean and tomato, flagella isolated from the parental strain were not effective, whereas LPS or pseudobactin did induce systemic resistance.

Antiprotease inactivation by Salmonella enterica released from infected macrophages

The mammalian serine protease plasmin, which has an important role in extracellular matrix degradation during cell migration, is regulated by the plasma antiprotease alpha(2)-antiplasmin (alpha(2)AP). The surface protease PgtE of Salmonella enterica serovar Typhimurium proteolytically inactivated alpha(2)AP. PgtE also activates the plasma zymogen plasminogen to plasmin, and bacteria expressing PgtE promoted degradation of extracellular matrix laminin in the presence of plasminogen and alpha(2)AP. alpha(2)AP inactivation was detected with the rough derivative of S. enterica 14028, but not with the smooth wild-type strain, suggesting that the O-antigen of lipopolysaccharide prevented contact of PgtE with the substrate molecule. After growth of S. enterica 14028 in murine J774A.1 macrophage-like cells, the infected cell lysate as well as bacteria from isolated Salmonella-containing vacuoles (SCVs) cleaved alpha(2)AP. Bacteria from SCVs produced an elevated level of PgtE and had a reduced O-antigen chain length. The lysate from S. enterica 14028-infected macrophages promoted formation of plasmin in the presence of alpha(2)AP, whereas plasmin formation by lysates from uninfected macrophages, or from macrophages infected with the pgtE-negative derivative of 14028, was inhibited by alpha(2)AP. Salmonella disseminates in the host within macrophages, which utilize plasmin for migration through tissue barriers. The results suggest that intracellular enhancement of PgtE activity in Salmonella may promote macrophage-associated proteolysis and cellular migration by altering the balance between host plasmin and alpha(2)AP.

Molecular genetics, biochemistry and biological role of Yersinia lipopolysaccharide.

Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria. The LPS molecule is composed of two biosynthetic entities: the lipid A--core and the O-polysaccharide (O-antigen). Most biological effects of LPS are due to the lipid A part, however, there is an increasing body of evidence also with Yersinia indicating that O-antigen plays an important role in effective colonization of host tissues, resistance to complement-mediated killing and in the resistance to cationic antimicrobial peptides that are key elements of the innate immune system. The biosynthesis of O-antigen requires numerous enzymatic activities and includes the biosynthesis of individual NDP-activated precursor sugars in the cytoplasm, linkage and sugar-specific transferases, O-unit flippase, O-antigen polymerase and O-chain length determinant. Based on this enzymatic mode of O-antigen biosynthesis LPS isolated from bacteria is a heterologous population of molecules; some do not carry any O-antigen while others that do have variation in the O-antigen chain lengths. The genes required for the O-antigen biosynthesis are located in O-antigen gene clusters that in genus Yersinia is located between the hemH and gsk genes. Temperature regulates the O-antigen expression in Y. enterocolitica and Y. pseudotuberculosis; bacteria grown at room temperature (RT, 22-25 degrees C) produce in abundance O-antigen while only trace amounts are present in bacteria grown at 37 degrees C. Even though the amount of O-antigen is known to fluctuate under different growth conditions in many bacteria very little detailed information is available on the control of the O-antigen biosynthetic machinery.

Structural characterization of the lipopolysaccharide O-polysaccharide antigen produced by Flavobacterium columnare ATCC 43622.

The structure of the antigenic O-chain polysaccharide of Flavobacterium columnare ATCC 43622, a Gram-negative bacterium that causes columnaris disease in warm water fish, was determined by high-field 1D and 2D NMR techniques, MS, and chemical analyses. The O-chain was shown to be an unbranched linear polymer of a trisaccharide repeating unit composed of 2-acetamido-2-deoxy-d-glucuronic acid (d-GlcNAcA), 2-acetamidino-2,6-dideoxy-l-galactose (l-FucNAm) and 2-acetamido-2,6-dideoxy-d-xylo-hexos-4-ulose (d-Sug) (1 : 1 : 1), having the structure: [structure: see text].

Proper expression of the O-antigen of lipopolysaccharide is essential for the virulence of Yersinia enterocolitica O:8 in experimental oral infection of rabbits

The O-antigen of lipopolysaccharide (LPS) is required for virulence in Yersinia enterocolitica serotype O:8. Here we evaluated the importance of controlling the O-antigen biosynthesis using an in vivo rabbit model of infection. Y. enterocolitica O:8 wild-type strain was compared to three mutants differing in the O-antigen phenotype: (i) the rough strain completely devoid of the O-antigen, (ii) the wzy strain that lacks the O-antigen polymerase (Wzy protein) and expresses LPS with only one repeat unit, and (iii) the wzz strain that lacks the O-antigen chain length determinant (Wzz protein) and expresses LPS without modal distribution of O-antigen chain lengths. The most attenuated strain was the wzz mutant. The wzz bacteria were cleared from the tissues by day 30, the blood parameters were least dramatic and histologically only immunomorphological findings were seen. The level of attenuation of the rough and the wzy strain bacteria was between the wild-type and the wzz strain. Wild-type bacteria were highly resistant to killing by polymorphonuclear leukocytes, the wzz strain bacteria were most sensitive and the rough and wzy strain bacteria were intermediate resistant. These results clearly demonstrated that the presence of O-antigen on the bacterial surface is not alone sufficient for full virulence, but also there is a requirement for its controlled chain length.

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

Novel Aeromonas hydrophila PPD134/91 genes involved in O-antigen and capsule biosynthesis.

The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells.

Helicobacter pylorifrom asymptomatic hosts expressing heptoglycan but lacking Lewis O-chains: Lewis blood-group O-chains may play a role inHelicobacter pyloriinduced pathology

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain --> core --> lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-alpha-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.

Analysis of the O-antigen chain length distribution during extracellular and intracellular growth of Shigella flexneri

This study has established that there were no changes in the general structure of LPS of Shigella flexneri M90T either when the bacteria grew free in the cytoplasm of the eucaryotic host cell or during extracellular growth in liquid LB medium at 37°C. In both cases there was a similar bi-modal O-antigen chain length distribution with the mean modal values between 1 and 2, and between 11 and 14 subunits. This suggests that the intracellular localization is not a significant stimulus perceived by Shigella to regulate the length of its O-side chains. However, when the bacteria grew under extracellular conditions (liquid medium) at 30°C, even though there were no changes in the modal values, the pattern of the O-antigen chain length distribution of LPS was different, with an increase in the amount of the long chains relative to the short chains.

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Isolation and characterization ofFrancisella novicidamutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.

Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65

The antigenic O-polysaccharide moiety of the lipopolysaccharide produced by Escherichia coli serotype O65 was investigated by composition, methylation, base hydrolysis, periodate oxidation, mass spectrometric methods, and by 1D and 2D NMR spectroscopy. The O-polysaccharide had [ α] D+108° (water) and is a high-molecular-weight unbranched linear polymer of repeating pentasaccharide units composed of 1:1:1:1:1 d-galacturonic acid ( d-GalA), d-galacturonamide ( d-GalANH 2), 2-acetamido-2-deoxy- d-glucose ( d-GlcNAc), 2-acetamido-2-deoxy- d-galactose ( d-GalNAc), and 3-acetamido-3,6-dideoxy- d-glucose ( d-Qui3NAc), and has the following structure:

Use of a murine O-antigen-specific monoclonal antibody to identify Acinetobacter strains of unnamed genomic species 13 Sensu Tjernberg and Ursing.

A monoclonal antibody against the O-antigenic polysaccharide chain of the lipopolysaccharide (LPS) of Acinetobacter strains belonging to the unnamed genomic species 13 Sensu Tjernberg and Ursing (13TU) was obtained after immunization of BALB/c mice with heat-killed bacteria and was characterized by enzyme immunoassay and Western blot analysis, by use of LPS and proteinase K-treated bacterial lysates, analyses in which the antibody was shown to be highly specific for the homologous antigen. In addition, when tested in dot and Western blots, reactivity was observed with 9 of 18 Acinetobacter strains of genomic species 13TU which had been isolated in Germany and Denmark; no reactivity was observed with strains of other genomic species, including the closely related genomic groups 1 (A. calcoaceticus), 2 (A. baumannii), and 3 (unnamed), or with other gram-negative bacteria. The antibody described here represents a convenient reagent for the simple, economical, and accurate differentiation of clinical isolates of genomic species 13TU from other Acinetobacter strains. Although the antibody does not identify all isolates of this genomic group, it is evident that it will be a useful reagent in the development of a serotyping scheme for clinical laboratories.

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.