Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.     

Lipopolysaccharide O-antigen chain length regulation in Pseudomonas aeruginosa serogroup O11 strain PA103

The Wzz proteins are important for determining the length of the O-antigen side chain attached to lipopolysaccharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wild-type PA103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzz1 protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.     

A Wzz (Cld) protein determines the chain length of K lipopolysaccharide in Escherichia coli O8 and O9 strains.

The modal distribution of O-antigen chain length is determined by the Wzz (Cld/Rol) protein in those cases in which it has been studied. The system of O-antigen synthesis in Escherichia coli serotypes O8 and O9 is different from that reported for most other bacteria, and chain length distribution is thought not to be determined by a Wzz protein. We report the existence in E. coli O8 and O9 strains of wzz genes which are very similar to and have sequences within the range of variation of those which determine the chain length of typical O antigens. We also find that wzz genes previously identified by their effect on O-antigen chain length, when cloned and transferred to O8 and O9 strains, affect the chain length of a capsule-related form of LPS, K(LPS). We conclude that in at least some O8 and O9 strains there is a wzz gene which controls the chain length of K(LPS) but has no effect on the O8 or O9 antigen.     

Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised patients and trauma victims and causes fatal lung infections in people with cystic fibrosis. This microorganism produces a number of virulence factors, one of which is lipopolysaccharide (LPS), which has been shown to mediate many biological effects including resistance to serum killing and phagocytosis. These biological activities have been correlated to the length of the O-polysaccharide and its distribution on the outer membrane. Wzz is responsible for regulation of the size distribution of the O-antigen. Wzz has been found to participate solely in the Wzy-dependent pathway for LPS biosynthesis, which produces heteropolymeric O-polysaccharide such as the B-band LPS of P. aeruginosa. Our laboratory has previously reported characterization of a Wzz protein encoded in the B-band O-antigen biosynthesis cluster of PAO1. The availability of the genome sequence of P. aeruginosa PAO1 has made it possible to identify a second functional Wzz protein (PA0938, Wzz2). Gene replacement was used to generate an unmarked wzz2delta knock-out and a wzz2delta/wzz1::Gm double knock-out. As expected, the wzz2delta strain produced LPS with modal length imparted by Wzz1, and the wzz2delta/wzz1::Gm strain produced LPS O-antigen with a non-modal (random) length. Both wzz1 and wzz2 from P. aeruginosa PAO1 were cloned and expressed with an N-terminal His6 tag. His6-Wzz1 and His6-Wzz2 were purified to near homogeneity by immobilized metal affinity chromatography (IMAC). These preparations were used to develop specific polyclonal antibodies against each of the proteins. In vivo protein cross-linking followed by Western immunoblotting indicated that Wzz1 forms dimers whereas Wzz2 forms octamers. By generation of a wzz2delta/rmlC double mutant and analysis of the LPS, we have made the novel observation that polymerization of modal chain length-distributed O-antigen occurred before ligation to the lipid A core. We have shown an association between the Wzz proteins and O-antigen polymer chains using immunoprecipitation with anti-O5 O-antigen monoclonal antibody MF15-4. Both Wzz1 and Wzz2 could be co-precipitated with O5 polymer.     

Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement

A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzzST and wzzfepE) or three heterologous (wzzO139 of Vibrio cholerae and wzzSF and wzzpHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.     

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.     

Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri

The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.     

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.     

Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.     

A plasmid-borne O-antigen chain length determinant and its relationship to other chain length determinants

Abstract We identify a function-controlling O antigen chain length for a plasmid-borne gene, cld _pHS-2 harboured by Flexneri strains of Escherichia coli known to cause reactive arthritis. The predicted amino acid sequence of the gene product is very similar to those of other cld genes and that of fepE , thought to be part of the enterobactin iron uptake system of E. coli . The predicted proteins are compared with rfb -associated chain length determinants as a family of related genes     

The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by o349 of the wec Gene Cluster of Escherichia coli K-12

The assembly of the phosphoglyceride-linked form of enterobacterial common antigen (ECA(PG)) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of o349 of the Escherichia coli K-12 wec gene cluster revealed that this locus encodes a Wzz protein that specifically modulates the chain length of ECA(PG) polysaccharides, and we have designated this locus wzz(ECA). The Wzz(ECA)-mediated modulation of ECA(PG) polysaccharide chains is the first demonstrated example of Wzz regulation involving a polysaccharide that is not linked to the core-lipid A structure of lipopolysaccharide.     

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.     

Molecular analysis of the O-antigen gene cluster of Escherichia coli O86:B7 and characterization of the chain length determinant gene (wzz)

Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.     

Conflicting roles for a cell surface modification in Salmonella

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O‐antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram‐negative bacteria. We determined that activation of the PmrA/PmrB two‐component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA‐activated gene wzz_st, which encodes the protein that determines long O‐antigen chain length and confers resistance to complement‐mediated killing. By contrast, this growth defect did not require the wzz_fepE gene, which controls production of very long O‐antigen, or other PmrA‐activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O‐antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer‐membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O‐antigen in its LPS to respond to the different conditions it faces during infection.     

Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.     

The Wzz (Cld) Protein in Escherichia coli: Amino Acid Sequence Variation Determines O-Antigen Chain Length Specificity

The O antigen is a polymer with a repeated unit. The chain length in most Escherichia coli strains has a modal value of 10 to 18 O units, but other strains have higher or lower modal values. wzz (cld/rol) mutants have a random chain length distribution, showing that the modal distribution is determined by the Wzz protein. Cloned wzz genes from E. coli strains with short (7 to 16), intermediate (10 to 18), and long (16 to 25) modal chain lengths were transferred to a model system, and their effects on O111 antigen were studied. The O111 chain length closely resembled that of the parent strains. We present data based on the construction of chimeric wzz genes and site-directed mutagenesis of the wzz gene to show that the modal value of O-antigen chain length of E. coli O1, O2, O7, and O157 strains can be changed by specific amino acid substitutions in wzz. It is concluded that the O-antigen chain length heterogeneity in E. coli strains is the result of amino acid sequence variation of the Wzz protein.     

Contribution of the lipopolysaccharide to resistance of Shigella flexneri 2a to extreme acidity

Shigella flexneri is endemic in most underdeveloped countries, causing diarrheal disease and dysentery among young children. In order to reach its target site, the colon, Shigella must overcome the acid environment of the stomach. Shigella is able to persist in this stressful environment and, because of this ability it can initiate infection following the ingestion of very small inocula. Thus, acid resistance is considered an important virulence trait of this bacterium. It has been reported that moderate acid conditions regulate the expression of numerous components of the bacterial envelope. Because the lipopolysaccharide (LPS) is the major component of the bacterial surface, here we have addressed the role of LPS in acid resistance of S. flexneri 2a. Defined deletion mutants in genes encoding proteins involved in the synthesis, assembly and length regulation of the LPS O antigen were constructed and assayed for resistance to pH 2.5 after adaptation to pH 5.5. The results showed that a mutant lacking O antigen was significantly more sensitive to extreme acid conditions than the wild type. Not only the presence of polymerized O antigen, but also a particular polymer length (S-OAg) was required for acid resistance. Glucosylation of the O antigen also contributed to this property. In addition, a moderate acidic pH induced changes in the composition of the lipid A domain of LPS. The main modification was the addition of phosphoethanolamine to the 1' phosphate of lipid A. This modification increased resistance of S. flexneri to extreme acid conditions, provide that O antigen was produced. Overall, the results of this work point out to an important role of LPS in resistance of Shigella flexneri to acid stress.     

Modulation of O‐antigen chain length by the wzz gene in Escherichia coli O157 influences its sensitivities to serum complement

Escherichia coli O157 strains belonging to a distinct lineage and expressing different O‐antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild‐type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS–PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157‐antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild‐type strain and the other wzz mutants (P < 0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains.     

Molecular and functional analysis of genes required for expression of group IB K antigens in Escherichia coli : characterization of the his-region containing gene clusters for multiple cell-surface polysaccharides

Escherichia coli group I capsular K antigens are found in two forms on the cell surface. The K_LPS form is linked to lipopolysaccharide lipid A core, whereas the high-molecular-weight capsular form is assembled independently of lipid A core. Subgroup IB K antigens are generally co-expressed with either the O8 or O9 antigen and, under the appropriate conditions, with the exopolysaccharide, colanic acid. To examine the relationships between the genetic loci and the synthetic pathways for these various cell-surface polymers, the gene cluster responsible for expression of a prototype group IB K antigen (serotype K40) was cloned and the flanking chromosomal regions characterized. Analysis of the six orf s within the cluster indicates features typical of Wzy (Rfc)-dependent O antigens. Synthesis of group IB K antigens is initiated by WecA (Rfe), a UDP-GlcNAc::undecaprenylphosphate GlcNAc-1-phosphate transferase, and the chain length of K40_LPS is determined by the wzz gene product. The his -region of the E . coli O8:K40 prototype is almost exclusively devoted to the expression of three different surface polysaccharides. The rfb _K40 cluster is located adjacent to the cps (colanic acid synthesis) and rfb _O8 (O8 antigen synthesis) loci in the gene order: his - rfb _O8/O9– wzz – ugd – gnd – rfb _K40– galF – cps . Thus, rfb _K40 is in the location occupied by other Wzy-dependent rfb gene clusters, and rfb _O8/O9 represents an additional locus.     

Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.