Rfb Gene Cluster Research Articles

Serotipado de Escherichia coli aisladas de pollos de engorde y determinación de resistencia a la Colistina

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically damaging to poultry industries Worldwide. E. coli strains of serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster that controls O antigen synthesis generally varies among different E. coli serotypes. In this study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared, and it was also aimed to search for Colistin resistance on a molecular basis. For the research, 200 swab samples were taken from 200 chickens suspected of colibacillosis in broiler poultry farms located in the vicinity of Aydın, İzmir, and Manisa Provinces in Turkey 2022. Bacterial growth was obtained from 92% of the samples, and microbiological analysis identified 108 (54%) Escherichia coli isolates. In addition, Klebsiella spp. was identified in 35 (17.5%) samples, Proteus spp. in 23 (11.5%), Pseudomonas spp. in 18 (9%), and no bacterial growth was observed in 16 (8%) samples. mcr-1 (309 bp) and mcr–2 (567 bp) genes responsible for Colistin resistance was investigated in plasmid DNA extracted from 108 E. coli isolates obtained in the study, using the PCR method. However, neither mcr-1 nor mcr–2 genes were detected in any of the samples. In conclusion, the allele-specific PCR method was found sensitive and applicable for APEC identification and multiple drug resistance emerged in E. coli strains isolated according to the antibiogram results.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Horizontal transfer of the rfb cluster in Leptospira is a genetic determinant of serovar identity.

Leptospira bacteria comprise numerous species, several of which cause serious disease to a broad range of hosts including humans. These spirochetes exhibit large intraspecific variation, resulting in complex tabulations of serogroups/serovars that crisscross the species classification. Serovar identity, linked to biological/clinical phenotypes, depends on the structure of surface-exposed LPS. Many LPS biosynthesis-encoding genes reside within the chromosomic rfb gene cluster. However, the genetic basis of intraspecies variability is not fully understood, constraining diagnostics/typing methods to cumbersome serologic procedures. We now show that the gene content of the rfb cluster strongly correlates with Leptospira serovar designation. Whole-genome sequencing of pathogenic L. noguchii, including strains of different serogroups, reveals that the rfb cluster undergoes extensive horizontal gene transfer. The rfb clusters from several Leptospira species disclose a univocal correspondence between gene composition and serovar identity. This work paves the way to genetic typing of Leptospira serovars, and to pinpointing specific genes within the distinct rfb clusters, encoding host-specific virulence traits. Further research shall unveil the molecular mechanism of rfb transfer among Leptospira strains and species.

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.

Pathological Investigation and Molecular Detection of Avian Pathogenic E. coli Serogroups in Broiler Birds

The objective of the present study the rfb gene clusters in avian pathogenic E. coli cardinal serotypes O1, O2 and O78 strains and to develop a multiplex polymerase chain reaction method for serotyping of the O-antigens. The multiplex polymerase chain reaction method was used for the identification of serotypes of APEC. The second part of the study was to study the pathological lesions caused by most prevalent sero group in experimentally infected broiler chicks. A total of 100 tissue samples (50 lungs and livers each) were collected from colibacillosis suspected broiler birds and subjected to isolation and identification of E. coli by conventional methods. Multiplex PCR was used for confirmation of three serogroups i.e., O1, O2 and O78. We found more O2 33% than O1 8% and O78 zero percent. These results suggested the prevalence of O2 sero group in our study. The prevalent sero group (O2) was experimentally inoculated in broiler birds at day 7 of their age. Lungs and liver samples from these experimentally infected birds were taken at days 14 and 21 of their age and subjected to histopathology. We found that there was hepatomegaly, coagulative necrosis, congestion and infiltration of inflammatory cells in infected livers. The lungs were congested and there were macrophages, lymphocytes and heterophils too. There was mostly hepatic form of colibacillosis with this infective strain.

Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

Development of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1, O2, O18 and O78 strains.

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.

PCR identification of Salmonella serogroups based on specific targets obtained by comparative genomics

Comparative genomic approaches provide abundant information to reveal the diversity among Salmonella serogroups. In a local genomic sequence database, twenty-five Salmonella whole genomic sequences were divided into 6 (A, B, C1, C2, D and others) serogroups for mining the DNA fragments specific for serogroups A through D. For each serogroup, a reference sequence was selected and split into 1000-bp fragments in silico to align against all the other genomic sequences to obtain one or more serogroup-specific fragments. As a result, 2, 6, 7, 10, and 7 specific fragments were found for A, B, C1, C2 and D serogroups, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 additional food isolates. The PCR results demonstrated good agreement with those from Salmonella serotyping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. Based on the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups.

The Escherichia coli O149 rfb gene cluster and its use for the detection of porcine E. coli O149 by real-time PCR

PCR or hybridization assays are widely used for the identification and detection of various Escherichia coli serogroups and serotypes. This study explored this approach for the detection of E. coli O149 in pigs, a dominant serogroup among those associated with porcine post-weaning diarrhea (PWD) worldwide. For this purpose, we sequenced the O antigen (rfb) gene cluster of a representative enterotoxigenic E. coli (ETEC) O149:H10 strain and identified a 2kbp region specific for the O149 rfb gene cluster. This region lacked similarity with sequences in GenBank, except for the rfb gene cluster of the human pathogen Shigella boydii O1 with which it shares 99% sequence identity. Southern blot hybridization with 91 E. coli strains belonging to 62 serogroups confirmed the specificity of the identified region. Restriction analysis of the rfb gene cluster of 38 geographically and chronologically diverse E. coli O149 strains with the endonucleases HindIII, PstI and XhoI demonstrated that this cluster is highly conserved across the serogroup O149. Analysis of the O antigen of a representative O149:H10 ETEC strain by nuclear magnetic resonance confirmed the identity of the O antigen structure in recent and older O149 ETEC strains. Finally, we developed a real-time PCR assay for the detection of E. coli O149 in pig fecal samples by targeting the part of the rfb sequence specific for this serogroup. This method was suitable for a sensitive qualitative detection of O149 E. coli in pig fecal samples after enrichment, but further investigations are needed to find a suitable method for a direct, reproducible and sensitive quantification of O149 E. coli in pig feces.

Application of PCR-based serogrouping of selected Salmonella serotypes in Malaysia

Differentiation of Salmonella enterica into its serogroups is important for epidemiological study. The objective of the study was to apply a multiplex PCR targeting serogroups A, B, C1, D, E and Vi-positive strains of Salmonella enterica commonly found in Malaysia. A separate H-typing multiplex PCR which identified flagellar antigen "a", "b" or "d" was also optimized to confirm clinical serotypes, S. Paratyphi A and S. Typhi. Sixty-seven laboratory Salmonella enterica strains were tested. Six sets of primers targeting defined regions of the O antigen synthesis genes (rfb gene cluster) and Vi antigen gene (viaB) were selected and combined into a multiplex PCR for O-grouping. Four primers (H-for, Ha-rev, Hb-rev and Hd-rev) were used in the second step multiplex PCR for H-typing. The optimized mPCR assays were further evaluated with 58 blind-coded Salmonella strains. The multiplex PCR results obtained showed 100% concordance to the conventionally typed serogroups. Validation with 58 blind coded Salmonella strains yield 100% accuracy and specificity. Based on this study, PCR serogrouping proved to be a rapid, alternative method for further differentiation of Salmonella enterica.

Genetic diversity among major endemic strains of Leptospira interrogans in China

BackgroundLeptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars).ResultsComparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification.ConclusionCGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.

Prevalence and numbers of Escherichia coli O157:H7 in minced beef and beef burgers from butcher shops and supermarkets in the Republic of Ireland

This study investigated the prevalence and numbers of Escherichia coli O157:H7 in minced beef and beef burgers in supermarkets and butcher shops in the Republic of Ireland. Fifteen samples were collected quarterly from each of 26 counties over a 13-month period. All samples ( n=1533) were (1) directly plated on SMAC, and (2) enriched in mTSB with novobiocin, extracted by immunomagnetic separation (IMS), plating onto SMAC-CT agar and finally confirmed by PCR. Overall, E. coli O157:H7 was recovered from 43 samples (2.80%) with counts ranging from <0.52–4.03 log 10 cfu g −1. Of the positive samples, 2.70% (32/1183) were purchased from supermarkets and 3.14% (11/350) from butcher shops. Only one product type (fresh unpacked burgers from supermarkets) was negative for E. coli O157:H7. Of the products containing the pathogen, fresh packaged burgers from supermarkets had the highest prevalence of 4.46% (7/157) while fresh unpackaged mince purchased from supermarkets had the lowest prevalence of 2.01% (6/299). Of the 43 isolates recovered, 41 possessed verotoxin-producing genes ( vt1 and vt2), E. coli attaching and effacing gene ( eaeA), haemolysin gene ( hlyA), 60-MDa plasmid or rfb gene cluster that encodes for the biosynthesis of the O-antigen (pO157) and flagellar H7 antigen encoding gene ( fliC h7). The remaining 2/43 isolates contained only one of the verotoxin-producing genes ( vt1 or vt2) and all the other genes named.

Detection of Salmonella enterica Somatic Groups C1 and E1 by PCR–Enzyme-Linked Immunosorbent Assay

Objectives of this study were to develop a PCR-based enzyme-linked immunosorbent assay (PCR-ELISA) for identification of Salmonella enterica somatic groups C1 and E1 and to evaluate this procedure along with a PCR-ELISA procedure for S. enterica somatic groups B, C2, and D in a masked study. Primers were selected from the rfb gene cluster, which is responsible for biosynthesis of O antigens of Salmonella lipopolysaccharide. Previously serogrouped Salmonella isolates (n = 169) were used to determine the sensitivity and specificity of the PCR-ELISA procedure. DNA from all isolates was amplified using the PCR procedure for selected somatic groups and amplified products were visualized on agarose gels, as well as subjected to the ELISA procedure. The PCR-ELISA technique correctly identified 97% of somatic group C1 and 87% of somatic group E1. The sensitivity of this procedure to correctly identify S. enterica somatic group C1 was 97% and 88% for somatic group E1. The specificity was 98% for both somatic groups C1 and E1. The PCR-ELISA techniques correctly identified 93% of Salmonella isolates belonging to somatic groups B, C1, C2, D, and E1. The overall sensitivity of this procedure to correctly identify S. enterica somatic groups was 96% and the specificity was 98%. Ninety-one percent of somatic group D, 92% of somatic group B, and 97% of somatic group C2 were identified correctly with this procedure. Results of this study indicate that the PCR-ELISA procedure is a rapid and accurate method for serogrouping Salmonella isolates. Utilization of the PCR-ELISA procedure for Salmonella serogrouping would aide in identification, surveillance, prevention, and control of Salmonella.

Histopathological study of rabbit intestinal mucosa infected with a hybrid strain of Shigella dysenteriae 1 carrying LPS biosynthesis genes of Salmonella enterica serovar typhimurium

The rfb gene cluster and the rfc gene of Salmonella enterica were introduced earlier into an invasive Shigella dysenteriae 1 strain by triparental cross. Antiserum was raised in rabbit against lipopolysaccharide isolated from the hybrid strain. Both the hybrid and the invasive S. dysenteriae 1 strain were found to have a titer of 1:2560 while for S. enterica, it was 1:640. Ligated ileal loops were prepared in rabbit, which were inoculated with 10 8 CFU ml −1 each of the hybrid strain, and invasive S. dysenteriae 1 strain used as positive control. Escherichia coli K12 was also used as a negative control. After 18 h, the fluid accumulation ratios were 0.2 and 1.6 for hybrid and invasive strains of S. dysenteriae 1, respectively. Rabbit intestinal mucosa infected with hybrid S. dysenteriae 1 strain showed the presence of intact villus tips and unruptured intestinal mucosa whereas total necrosis of intestinal mucosa and villi was observed in the S. dysenteriae 1-infected region.

Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae.

Despite over 100 years of study, the intestinal pathogen Vibrio cholerae still causes epidemic disease in areas of the world where there is poor sanitation. While cholera toxin and the toxin-coregulated pilus (TCP) are known to be essential for full virulence, the role that other factors play has remained ill-defined. Herein, we describe a large-scale signature-tagged mutagenesis (STM) screen utilizing 100 pools of 96 mutants each to identify factors involved in colonization of the infant mouse small intestine. A total of 164 mutants representing transposition events into 95 different open reading frames were shown to be recovered at greatly reduced numbers from the infant mouse model. Analysis of the sites of insertion revealed multiple independent mutations within the rfb gene cluster, needed for synthesis of lipopolysaccharide (LPS), and the tcp gene cluster, needed for synthesis of the TCP. More importantly, in addition to these previously known colonization factors, we identified many genes whose activity in colonization was not previously appreciated. These can be divided into a number of functional groups, which include production of factors involved in metabolic activities, regulation of cellular processes, transport, adaptation to stress and unknown functions. In addition, we describe the reiterative use of STM, whereby colonization-defective mutants were assembled into virulence-attenuated pools (VAPs), which were used to begin to reveal roles that the identified virulence factors play in the infection process. Nine new factors were shown to be crucial for the V. cholerae acid tolerance response, which has previously been hypothesized to be important for epidemic spread of cholera. Competition assays of these nine acid tolerance response (ATR)-defective mutants revealed that mutations in gshB, hepA and recO result in a 1000-fold reduction in colonization.

Two functional O-polysaccharide polymerase wzy ( rfc) genes are present in the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum

Defined regions of the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum were introduced into a mutated derivative of this strain that lacks O-polysaccharide polymerase activity. Three different kinds of assays performed on the various transformants all indicate that two functional wzy ( rfc) genes reside within the Group E1 Salmonella rfb gene cluster. The product of ORF9.6, positioned near the center of the rfb gene cluster, joins O-polysaccharide repeat units together by α-glycosidic linkages to produce antigen O10, the major serological determinant of Group E1 S. enterica. The product of ORF17.4, positioned at the downstream end of the rfb gene cluster, can join repeat units together by β-glycosidic linkages to produce antigen O15, the major serological determinant of Group E2 S. enterica.

Two functional O-polysaccharide polymerase wzy (rfc) genes are present in the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum.

Defined regions of the rfb gene cluster of Group E1 Salmonella enterica serovar Anatum were introduced into a mutated derivative of this strain that lacks O-polysaccharide polymerase activity. Three different kinds of assays performed on the various transformants all indicate that two functional wzy (rfc) genes reside within the Group E1 Salmonella rfb gene cluster. The product of ORF9.6, positioned near the center of the rfb gene cluster, joins O-polysaccharide repeat units together by alpha-glycosidic linkages to produce antigen O10, the major serological determinant of Group E1 S. enterica. The product of ORF17.4, positioned at the downstream end of the rfb gene cluster, can join repeat units together by beta-glycosidic linkages to produce antigen O15, the major serological determinant of Group E2 S. enterica.

Shigella dysenteriae type 1 carrying LPS biosynthesis genes of Salmonella typhimurium affects both Invasive Plasmid AntigenH (IpaH) secretion and invasion

A Shigella dysenteriae 1 strain isolated from an epidemic in West Bengal, India. The strain contained six plasmids including a large virulence plasmid. A plasmid, pPR1347 carrying both the rfb gene cluster and the rfc gene of Salmonella typhimurium has been transferred to this invasive Shigella dysenteriae 1 strain by triparental cross with a very low frequency. Only five stable (100%) clones were isolated after examining several thousand colonies. All five transconjugants were Sereny negative and were unable to invade the HeLa cells. Transconjugants exhibited strong cross reactivity with S. dysenteriae 1 antisera but they showed weak reaction with Salmonella typhimurium antisera. Plasmid profiles of the transconjugants were unaltered as compared with the wild type strain except for the presence of pPR 1347. The transconjugants regained their invasive property after elimination (curing) of pPR 1347. However, Shigella-infected convalescent phase serum was able to detect IpaABCD proteins from whole cell lysate and culture supernatant of transconjugants and cured (pPR 1347) transconjugants. A 60 kDa IpaH protein was not secreted into the culture supernatant by the transconjugants. Synthesis of lipopolysaccharides (LPS) of the hybrid strains was increased within the region of 43 to 67 kDa in comparison with the wild type S. dysenteriae 1 strains.

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 orfs 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 K40LPS 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 rfbK40 cluster is located adjacent to the cps (colanic acid synthesis) and rfbO8 (O8 antigen synthesis) loci in the gene order: his-rfbO8/O9-wzz-ugd-gnd-rfbK40-galF-cps. Thus, rfbK40 is in the location occupied by other Wzy-dependent rfb gene clusters, and rfbO8/O9 represents an additional locus.

Evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella

In order to clarify the evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharides in Escherichia coli and Klebsiella, we studied the DNA sequence of the boundary region between the rfb and his genes in a series of strains possessing mannose homopolymer as O-specific polysaccharide. All had a characteristic gene organization carrying no gene between the rfb and his genes. Further, the recombination event was suggested to occur at the same site of the hisI gene in those strains. It was suggested that there was a close evolutionary relationship among rfb gene clusters synthesizing mannose homopolymer as O-specific polysaccharide in E. coli and Klebsiella.