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     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

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.     

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.     

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.     

Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the ipaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 NotI restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence-associated chromosomal loci.     

Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D

We report the cloning and mapping of the entire rfb gene cluster of a group C2 Salmonella strain. Comparison with the rfb region of group B strain LT2 and group D strain Ty2 reveals an 11.8 kb central region of limited similarity flanked by regions of high similarity. The genes from the central region confer a group C2 O-antigen structure on a Salmonella LT2 partial delete strain. The significance of this region in relation to function and evolutionary origin is discussed. We also report evidence for the existence of an O-antigen chain-length determinant in Escherichia coli K12 and propose a model for a possible mechanism by which a preferred chain length is determined.     

Reduced amounts of LPS affect both stress tolerance and virulence of Salmonella enterica serovar Dublin

Signature-tagged mutagenesis (STM) is a widely used technique for identification of virulence genes in bacterial pathogens. While this approach often generates a large number of mutants with a potential reduction in virulence a major task is subsequently to determine the mechanism by which the mutations influence virulence. Presently, we have characterised a Salmonella enterica serovar Dublin STM mutant that, in addition to having reduced virulence, was also impaired when growing under various stress conditions. The mutation mapped to the manC (rfbM) gene of the O-antigen gene cluster involved in O-antigen synthesis. The O-antigen is a component of the lipopolysaccharide (LPS) forming a unique constituent of the outer membrane of Gram-negative bacteria. While mutations in the O-antigen genes usually eliminate the entire O-antigen side chain we found that the transposon mutant produced intact O-antigen, however, the mutation reduced the amount of LPS.     

PCR test systems for genetic identification of enterohemorrhagic Escherichia coli

The analysis of modern data on the development of amplification test systems for the gene indication of enterohemorrhagic E. coli (EHEC) is presented. In this work the emphasis is laid on the importance of using specific primers whose nucleotide sequence is closely linked with genes controlling the key factors of EHEC pathogenicity; these factors include the determinants of the synthesis of adhesins and invasins (bfp, eae, tir), shiga-like toxins (stx1, stx2), enterohemolysin (ehx), serine protease (epsA) and specific LPS of O-antigen (rfb). The problem of using primers whose sequence is not linked with virulence genes, but which may also be used for the gene indication of E. coli O157:H7 (uid, fliC) is discussed.     

Characterization of the rfc region of Shigella flexneri.

The O antigen of the Shigella flexneri lipopolysaccharide (LPS) is an important virulence determinant and immunogen. We have isolated S. flexneri mutants which produce a semi-rough LPS by using an O-antigen-specific phage, Sf6c. Western immunoblotting was used to show that the LPS produced by the semi-rough mutants contained only one O-antigen repeat unit. Thus, the mutants are deficient in production of the O-antigen polymerase and were termed rfc mutants. Complementation experiments were used to locate the rfc adjacent to the rfb genes on plasmid clones previously isolated and containing this region (D. F. Macpherson, R. Morona, D. W. Beger, K.-C. Cheah, and P. A. Manning, Mol. Microbiol 5:1491-1499, 1991). A combination of deletions and subcloning analysis located the rfc gene as spanning a 2-kb region. Insertion of a kanamycin resistance cartridge into a SalI site in this region inactivated the rfc gene. The DNA sequence of the rfc region was determined. An open reading frame spanning the SalI site was identified and encodes a protein with a predicted molecular mass of 43.7 kDa. The predicted protein is highly hydrophobic and showed little sequence homology with any other protein. Comparison of its hydropathy plot with that of other Rfc proteins from Salmonella enterica (typhimurium) and Salmonella enterica (muenchen) revealed that the profiles were similar and that the proteins have 12 or more potential membrane-spanning segments. A comparison of the S. flexneri rfc gene and protein product with other rfc and rfc-like proteins revealed that they have a similarly low percentage of G + C content and have similar codon usage, and all have a high percentage of rare codons. An attempt to identify the S. flexneri Rfc protein was unsuccessful, although proteins encoded upstream and downstream of the rfc gene could be identified. Examination of the distribution of rare or minor codons in the rfc gene revealed that it has several minor codons within the first 25 amino acids. This is in contrast to the upstream gene rfbG, which also has a high percentage of rare codons but whose gene product could be detected. The positioning of the rare codons in the rfc gene may restrict translation and suggests that minor isoaccepting tRNA species may be involved in translational regulation of rfc expression. The low percentage of G + C content of rfc genes may be a consequence of the selection pressure to maintain this form of control.     

O-antigen structure in a virulent strain of Aeromonas hydrophila

Abstract Lipopolysaccharide (LPS) was isolated from a strain of Aeromonas hydrophila which had displayed serological, bacteriophage attachment and virulence properties similar to those found in strains of Aeromonas salmonicida . The structure of the O-antigen was determined and had many points of similarity with that previously elucidated for the O-antigen of A. salmonicida . Methylation analysis, chromium trioxide oxidation and ¹H-n.m.r. were used to confirm that the repeating unit of the O-chain had the following structure:
     

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.     

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.     

Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O-antigen specificity

The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O-antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O-antigen biosynthesis were generated from separate cosmid clones by deletion with Clal. These plasmids expressed O-antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These plasmids also mediated O-antigen expression in an E. coli K12 rfb-delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O-antigen biosynthesis to be defined to a maximum of 11 kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O-antigen unit. Analysis of plasmid-encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM-2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.     

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.     

Synthesis of the flavonoid-induced lipopolysaccharide of Rhizobium Sp. strain NGR234 requires rhamnosyl transferases encoded by genes rgpF and wbgA

In the presence of flavonoids, Rhizobium sp. strain NGR234 synthesizes a new lipopolysaccharide (LPS), characterized by a rhamnan O-antigen. The presence of this rhamnose-rich LPS is important for the establishment of competent symbiotic interactions between NGR234 and many species of leguminous plants. Two putative rhamnosyl transferases are encoded in a cluster of genes previously shown to be necessary for the synthesis of the rhamnose-rich LPS. These two genes, wbgA and rgpF, were mutated. The resulting mutant strains synthesized truncated rough LPS species rather than the wild-type rhamnose-rich LPS when grown with flavonoids. Based on the compositions of these purified mutant LPS species, we inferred that RgpF is responsible for adding the first one to three rhamnose residues to the flavonoid-induced LPS, whereas WbgA is necessary for the synthesis of the rest of the rhamnan O-antigen. The NGR234 homologue of lpsB, which, in other bacteria, encodes a glycosyl transferase acting early in synthesis of the core portion of LPS, was identified and also mutated. LpsB was required for all the LPS species produced by NGR234, in the presence or absence of flavonoids. Mutants (i.e., of lpsB and rgpF) that lacked any portion of the rhamnan O-antigen of the induced LPS were severely affected in their symbiotic interaction with Vigna unguiculata, whereas the NGR?wbgA mutant, although having very few rhamnose residues in its LPS, was able to elicit functional nodules.     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.