The Flag-2 locus, an ancestral gene cluster, is potentially associated with a novel flagellar system from Escherichia coli

Escherichia coli K-12 possesses two adjacent, divergent, promoterless flagellar genes, fhiA-mbhA, that are absent from Salmonella enterica. Through bioinformatics analysis, we found that these genes are remnants of an ancestral 44-gene cluster and are capable of encoding a novel flagellar system, Flag-2. In enteroaggregative E. coli strain 042, there is a frameshift in lfgC that is likely to have inactivated the system in this strain. Tiling path PCR studies showed that the Flag-2 cluster is present in 15 of 72 of the well-characterized ECOR strains. The Flag-2 system resembles the lateral flagellar systems of Aeromonas and Vibrio, particularly in its apparent dependence on RpoN. Unlike the conventional Flag-1 flagellin, the Flag-2 flagellin shows a remarkable lack of sequence polymorphism. The Flag-2 gene cluster encodes a flagellar type III secretion system (including a dedicated flagellar sigma-antisigma combination), thus raising the number of distinct type III secretion systems in Escherichia/Shigella to five. The presence of the Flag-2 cluster at identical sites in E. coli and its close relative Citrobacter rodentium, combined with its absence from S. enterica, suggests that it was acquired by horizontal gene transfer after the former two species diverged from Salmonella. The presence of Flag-2-like gene clusters in Yersinia pestis, Yersinia pseudotuberculosis, and Chromobacterium violaceum suggests that coexistence of two flagellar systems within the same species is more common than previously suspected. The fact that the Flag-2 gene cluster was not discovered in the first 10 Escherichia/Shigella genome sequences studied emphasizes the importance of maintaining an energetic program of genome sequencing for this important taxonomic group.     

Detection and characterization of the flagellar master operon in the four Shigella subgroups.

Strains in the genus Shigella are nonmotile, but they retain some cryptic flagellar operons whether functional or defective (A.Tominaga, M. A.-H. Mahmoud, T. Mukaihara, and M. Enomoto, Mol. Microbiol. 12:277-285, 1994). To disclose the cause of motility loss in shigellae, the presence or defectiveness of the flhD and flhC genes, composing the master operon whose mutation causes inactivation of the entire flagellar regulon, was examined in the four Shigella subgroups. The flhD operon cloned from Shigella boydii and Shigella sonnei can activate, though insufficiently, the regulon in the Escherichia coli flhD or flhC mutant background. The clone from Shigella dysenteriae has a functional flhD gene and nonfunctional flhC gene, and its inactivation has been caused by the IS1 element inserted in its 5' end. The operon of Shigella flexneri is nonfunctional and has suffered an IS1-insertion mutation at the 5' end of the flhD gene. Comparison of restriction maps indicates that only the central 1.8-kb region, including part of the flhC gene and its adjacent mot operon, is conserved among the four Shigella subgroups as well as in E. coli, but in Salmonella typhimurium the whole map is quite different from the others. Motility loss in shigellae is not attributable to genetic damage in the master operon of a common ancestor, but it occurs separately in respective ancestors of the four subgroups, and in both S. dysenteriae and S.flexneri IS1 insertion in the master operon might be the primary cause of motility loss.     

The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon

The nucleotide sequence of the biotin (bio) biosynthetic operon of Escherichia coli has been determined. The 5.8-kilobase region contains the five biotin operon genes, bioA, B, F, C, and D. and an open reading frame of unknown function. The operon is negatively regulated and divergently transcribed from a control region between the bioA and bioB genes. The product of the bioA gene, 7,8-diaminopelargonic acid aminotransferase, was discovered to be related to ornithine aminotransferase. The product of the bioF gene, 7-keto-8-aminopelargonic acid synthetase, was found to be similar to 5-aminolevulinic acid synthetase.     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.     

Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli.

The fliL operon of Escherichia coli contains seven genes that are involved in the biosynthesis and functioning of the flagellar organelle. DNA sequences for the first three genes of this operon have been reported previously. A 2.2-kb PstI restriction fragment was shown to complement known mutant alleles of the fliO, fliP, fliQ, and fliR genes, the four remaining genes of the fliL operon. Four open reading frames were identified by DNA sequence analysis and correlated to their corresponding genes by complementation analysis. These genes were found to encode very hydrophobic polypeptides with molecular masses of 11.1, 26.9, 9.6, and 28.5 kDa for FliO, FliP, FliQ, and FliR, respectively. Analysis of recombinant plasmids in a T7 promoter-polymerase expression system enabled us to identify three of the four gene products. On the basis of DNA sequence analysis and in vivo protein expression, it appears that the fliP gene product is synthesized as a precursor protein with an N-terminal signal peptide of 21 amino acids. The FliP protein was homologous to proteins encoded by a DNA sequence upstream of the flaA gene of Rhizobium meliloti, to a gene involved in pathogenicity in Xanthomonas campestris pv. glycines, and to the spa24 gene of the Shigella flexneri. The latter two genes encode proteins that appear to be involved in protein translocation, suggesting that the FliP protein may have a similar function.     

Molecular basis of the indole-negative reaction in Shigella strains: extensive damages to the tna operon by insertion sequences

The molecular basis of the loss of tryptophan utilization (indole-negative phenotype) of Shigella strains, in effect clones of Escherichia coli, was investigated. Analysis of the tna operon of 23 Shigella strains representing each of the indole-negative serotypes revealed that insertion sequence-mediated insertion and/or deletions damaged the tna operon, leading to inability to convert tryptophan to indole. These events differ for cluster 1, cluster 3, and the outlier Shigella strains, confirming our previous observation of independent origins of these lineages from within E. coli. Parallel loss of the trait and prevalence of indole-negative strains suggest that the trait is deleterious in Shigella strains and advantages those without it.     

Two novel flagellar components and H-NS are involved in the motor function of Escherichia coli

A mutation in H-NS results in non-flagellation of Escherichia coli due to a reduced expression of the flhDC master operon. We found that the hns-negative strain restored its flagellation in the presence of flhDC, although the resulting strain was still non-motile. Since the intracelluar levels of motor components MotA, MotB, and FliG in the Deltahns strain were unaltered, the non-motility indicates that H-NS affects flagellar function as well as biogenesis. We obtained an insertion in ycgR, a putative gene encoding a protein of 244 amino acid residues, which suppresses the motility defect of hns-deficient cells. The abnormally low swimming speed of hns mutant cells was fully restored by an insertion in ycgR, as assessed with computer-assisted motion analysis. A similar suppressor phenotype was observed with a multicopy expression of yhjH, a putative gene encoding a polypeptide of 256 amino acid residues. Since the flagella of most hns-deficient cells were not rotating, except a few with reduced speed, the suppression appears to increase the number of rotating flagella as observed with tethered bacteria. The ycgR and yhjH genes contain the consensus sequence found among the class III promoters of the flagellar regulon, and their expression monitored with a lacZ fusion requires FlhDC. These findings suggest that ycgR and yhjH, together with H-NS, are involved in the motor function and constitute new members of the flagellar regulon.     

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.     

Molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) pathogenicity islands in F165-positive E. coli strain from a diseased animal

Septicemic Escherichia coli 4787 (O115: K-: H51: F165) of porcine origin possess gene clusters related to extraintestinal E. coli fimbrial adhesins. This strain produces two fimbriae: F165(1) and F165(2). F165(1) (Prs-like) belongs to the P fimbrial family, encoded by foo operon and F165(2) is a F1C-like encoded by fot operon. Data from this study suggest that these two operons are part of two PAIs. PAI I(4787) includes a region of 20 kb, which not only harbors the foo operon but also contains a potential P4 integrase gene and is located within the pheU tRNA gene, at 94 min of the E. coli chromosome. PAI II(4787) includes a region of over 35 kb, which harbors the fot operon, iroBCDEN gene clusters, as well as part of microcin M genes and nonfunctional mobility genes. PAI II(4787) is found between the proA and yagU at 6 min of the E. coli chromosome.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

The selC-associated SHI-2 pathogenicity island of Shigella flexneri

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non-pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI-2 (Shigella island 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI-2. SHI-2-hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI-2 may allow Shigella survival in stressful environments, such as those encountered during infection.