pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2.

Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34. Homologous gene transfer in P. fluorescens 6.2 and A. eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization. Loci involved in autotrophic growth were among those transferred in A. eutrophus. In heterospecific matings, markers were transferred from P. fluorescens to A. eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A. eutrophus to P. fluorescens, and from Erwinia carotovora subsp. chrysanthemi to A. eutrophus. Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid. When S. typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113. R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu. R-prime plasmids could be transferred easily into different recipients and were used in complementation studies. PstI restriction digests of four R-prime plasmids carrying P. fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Restoration of phenotype in Escherichia coli auxotrophs by pULB113-mediated mobilisation from methylotrophic bacteria

The plasmid pULB113 has been transferred to the facultative methylotrophs, Methylobacterium AM1 and Methylobacterium extorquens and to the obligate methanotroph Methylosinus trichosporium OB3b. Subsequent transfer from the methanotroph to the facultative methylotrophs and from the methanotroph and the facultative methylotrophs to Escherichia coli K-12 strains has been observed. In crosses from M. extorquens to restriction-deficient recombination-impaired E. coli strains, restoration of phenotype to auxotrophs was demonstrated and R-primes isolated from these recipients could transform auxotrophic strains to prototrophy.     

Mobilization of the genes for photosynthesis from Rhodopseudomonas capsulata by a promiscuous plasmid.

Plasmid pBLM2, a derivative of RP1 with enhanced chromosome mobilization activity in Rhodopseudomonas capsulata, was isolated by screening rare exconjugant clones for sex factor activity. pBLM2 mobilized all known genes affecting photosynthesis as well as chromosomal genes for streptomycin and rifampin resistance and tryptophan and cytochrome biosynthesis. Tight linkage was exhibited among the genes affecting photosynthesis. The frequency of successful transfer of chromosomal markers reached 6 X 10(-4) per donor cell. R-primes were occasionally formed during conjugation, and a number of R-primes bearing the genes for photosynthesis were isolated by screening R. capsulata exconjugants with complementation phenotypes for the ability to transmit plasmid-borne R. capsulata genes to Escherichia coli cells. These R-primes were unstable in R. capsulata, but stable in E. coli or Pseudomonas fluorescens. Complementation and recombination events that occurred upon introduction of R-primes into R. capsulata mutants with altered photosynthetic apparatuses could be visualized as variations in colony pigmentation. Each R-prime studied complemented all known types of mutation affecting the differentiation of the photosynthetic apparatus, and no other R. capsulata gene was identified on those plasmids. The R. capsulata genes borne on the R-primes were not functional in E. coli or P. fluorescens.     

The stability of O-antigen plasmid is determined by a chromosomal region of Shigella dysenteriae 1

It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species of Shigella. In Shigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. In Shigella dysenteriae 1, a 10 kilobase (kb) plasmid is required for O-antigen production. Shigella dysenteriae 1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained in Escherichia coli K-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses of Shigella dysenteriae 1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique to Shigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which the his locus has been substituted with the histidine region of an E. coli K-12 chromosome. The S. dysenteriae IDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in an in vitro assay. Thus the 10-kb plasmid of Shigella dysenteriae 1 is required for O-antigen synthesis but not for cell invasion.     

In vivo cloning of Erwinia carotovora genes involved in the catabolism of hexuronates

Using the RP4::mini-Mu pULB113 plasmid, an RP4 derivative carrying a deleted Mu prophage which allows the plasmid to pick up any chromosomal DNA segment to form R' plasmids, we cloned all of the genes of Erwinia carotovora involved in the catabolism of the hexuronates and in the transport of these substrates. With the R' plasmids we isolated, we performed complementation analysis and found that, in the Erwinia carotovora strain we used, the genes involved in the catabolism of the hexuronates are clustered in four regions of the chromosome. This genetic organization is compared with that of Escherichia coli K-12.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes.

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.     

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.     

Influence of different rol gene products on the chain length of Shigella dysenteriae type 1 lipopolysaccharide O antigen expressed by Shigella flexneri carrier strains.

Introduction of the rol genes of Shigella dysenteriae 1 and Escherichia coli K-12 into Shigella flexneri carrier strains expressing the heterologous S. dysenteriae type 1 lipopolysaccharide resulted in the formation of longer chains of S. dysenteriae 1 O antigen. In bacteria producing both homologous and heterologous O antigen, this resulted in a reduction of the masking of heterologous O antigen by homologous lipopolysaccharide and an increased immune response induced by intraperitoneal immunization of mice by recombinant bacteria. The rol genes of S. dysenteriae 1 and E. coli K-12 were sequenced, and their gene products were compared with the S. flexneri Rol protein. The primary sequence of S. flexneri Rol differs from both E. coli K-12 and S. dysenteriae 1 Rol proteins only at positions 267 and 270, which suggests that this region may be responsible for the difference in biological activities.     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene escape model: transfer of heavy metal resistance genes from Escherichia coli to Alcaligenes eutrophus on agar plates and in soil samples.

Conjugal transfer from Escherichia coli to Alcaligenes eutrophus of the A. eutrophus genes coding for plasmid-borne resistance to cadmium, cobalt, and zinc (czc genes) was investigated on agar plates and in soil samples. This czc fragment is not expressed in the donor strain, E. coli, but it is expressed in the recipient strain, A. eutrophus. Hence, expression of heavy metal resistance by cells plated on a medium containing heavy metals represents escape of the czc genes. The two plasmids into which this DNA fragment has been cloned previously and which were used in these experiments are the nonconjugative, mobilizable plasmid pDN705 and the nonconjugative, nonmobilizable plasmid pMOL149. In plate matings at 28 to 30 degrees C, the direct mobilization of pDN705 occurred at a frequency of 2.4 x 10(-2) per recipient, and the mobilization of the same plasmid by means of the IncP1 conjugative plasmids RP4 or pULB113 (present either in a third cell [triparental cross] or in the recipient strain itself [retromobilization]) occurred at average frequencies of 8 x 10(-4) and 2 x 10(-5) per recipient, respectively. The czc genes cloned into the Tra- Mob- plasmid pMOL149 were transferred at a frequency of 10(-7) to 10(-8) and only by means of plasmid pULB113. The direct mobilization of pDN705 was further investigated in sandy, sandy-loam, and clay soils. In sterile soils, transfer frequencies at 20 degrees C were highest in the sandy-loam soil (10(-5) per recipient) and were enhanced in all soils by the addition of easily metabolizable nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of bacterial DNA in vivo using a transposable R6K ori and a P1vir phage.

A new method of cloning in vivo using the P1vir phage and transposon Tn5-rpsL oriR6K was developed. The method relies upon recircularization of transducing DNA containing a transposon insertion in a recombination-deficient strain of Escherichia coli K-12 and subsequent stable replication of the recircularized DNA. Using this method, we were able to clone in vivo the chromosomal region located between approximately 7.1 and 9.2 min on the E. coli K-12 map in a 95-kb plasmid.     

Analysis of R-primes demonstrates that genes for broad host range nodulation of Rhizobium strain NGR234 are dispersed on the Sym plasmid

Summary R-prime plasmids carrying regions of the symbiotic (Sym) plasmid of the broad host range Rhizobium strain NGR234 were isolated in intergeneric matings with Escherichia coli K12. Three R-primes carrying approximately 180 kb (pMN23), 220 kb (pMN31) and 330 kb (pMN49) of Sym DNA were characterized in more detail. Restriction enzyme analysis and hybridization studies showed that these R-primes carried large overlapping regions of the Sym plasmid, and had the symbiotic genes (two copies of nifH, D and K; nodA, B, C and D; region II; host specific nodulation (hsn) genes) located over half of the 470 kb Sym plasmid. Only the largest of these R-primes (pMN49) contained the complete nodulation host range of the original parent strain NGR234. This broad host range was shown to be present on plasmid pMN49 by being expressed in Agrobacterium tumefaciens strain A136. Furthermore the R-prime plasmids were shown to contain different regions of distinctive host specific nodulation (hsn) for tropical legume infection and for the nodulation of the non-legume Parasponia. Nodulation of soybeans, however, required an additional region that was not essential for the nodulation of other tropical legumes. Strain NGR234 was also found to nodulate the stem and roots of the tropical legume Sesbania rostrata at a very low efficiency. However, the R-prime mini Sym plasmid constructions enabled a greater efficiency of nodulation of Sesbania rostrata to occur.     

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.