Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.     

Variation between pathogenic serovars within Salmonella pathogenicity islands

Although four of the five Salmonella pathogenicity islands (SPIs) have been characterized in detail for Salmonella enterica serovar Typhimurium, and the fifth has been characterized for Salmonella enterica serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of S. enterica. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the avrA gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the pagJ and pagK genes between pipC and pipD. In SPI-3 the genes sugR and rhuM were deleted in most serovars and in some were replaced by sequences that were very similar to either the Escherichia coli fimbrial operon, flanked by two distinct insertion sequence elements, or to the E. coli retron phage PhiR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into S. enterica and that in some cases the same event has occurred in multiple lineages of S. enterica. Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic Salmonella serovars.     

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.     

Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract

Salmonella enterica subsp. enterica serovar Newport resistant to the extended-spectrum cephalosporins (ESCs) and other antimicrobials causes septicemic salmonellosis in humans and animals and is increasingly isolated from humans, animals, foods, and environmental sources. Mechanisms whereby serovar Newport bacteria become resistant to ESCs and other classes of antimicrobials while inhabiting the intestinal tract are not well understood. The present study shows that 25.3% of serovar Newport strains isolated from the turkey poult intestinal tract after the animals were dosed with Escherichia coli harboring a large conjugative plasmid encoding the CMY-2 beta-lactamase and other drug resistance determinants acquired the plasmid and its associated drug resistance genes. The conjugative plasmid containing the cmy-2 gene was transferred not only from the donor E. coli to Salmonella serovar Newport but also to another E. coli serotype present in the intestinal tract. Laboratory studies showed that the plasmid could be readily transferred between serovar Newport and E. coli intestinal isolates. Administration of a single dose of ceftiofur, used to prevent septicemic colibacillosis, to 1-day-old turkeys did not result in the isolation of ceftiofur-resistant E. coli or Salmonella serovar Newport. There was a remarkable association between serotype, drug resistance, and plasmid profile among the E. coli strains isolated from the poults. This study shows that Salmonella serovar Newport can become resistant to ESCs and other antibiotics by acquiring a conjugative drug resistance plasmid from E. coli in the intestines.     

A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA

Salmonella enterica serovar blegdam has a restriction and modification system encoded by genes linked to serB . We have cloned these genes, putative alleles of the hsd locus of Escherichia coli  K-12, and confirmed by the sequence similarities of flanking DNA that the hsd genes of S. enterica serovar blegdam have the same chromosomal location as those of E. coli K-12 and Salmonella enterica serovar typhimurium LT2. There is, however, no obvious similarity in their nucleotide sequences, and while the gene order in S. enterica serovar blegdam is serB hsdM , S and R , that in E. coli K-12 and S. enterica serovar typhimurium LT2 is serB hsdR , M and S . The hsd genes of S. enterica serovar blegdam identify a third family of serB -linked hsd genes (type ID). The polypeptide sequence predicted from the three hsd genes show some similarities (18–50% identity) with the polypeptides of known and putative type I restriction and modification systems; the highest levels of identity are with sequences of Haemophilus influenzae Rd. The HsdM polypeptide has the motifs characteristic of adenine methyltransferases. Comparisons of the HsdR sequence with those for three other families of type I systems and three putative HsdR polypeptides identify two highly conserved regions in addition to the seven proposed DEAD-box motifs.     

Comparison of Salmonella enterica serovar Typhimurium LT2 and non-LT2 salmonella genomic sequences, and genotyping of salmonellae by using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX)

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.     

Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain

The O antigen of Escherichia coli O111 is identical in structure to that of Salmonella enterica serovar adelaide. Another O-antigen structure, similar to that of E. coli O111 and S. enterica serovar adelaide is found in both E. coli O55 and S. enterica serovar greenside. Both O-antigen structures contain colitose, a 3,6 dideoxyhexose found only rarely in the Enterobacteriaceae. The O-antigen structure is determined by genes generally located in the rfb gene cluster. We cloned the rfb gene cluster from an E. coli O111 strain (M92), and the clone expressed O antigen in both E. coli K-12 and a K-12 strain deleted for rfb. Lipopolysaccharide analysis showed that the O antigen produced by strains containing the cloned DNA is polymerized. The chain length of O antigen was affected by a region outside of rfb but linked to it and present on some of the plasmids containing rfb. The rfb region of M92 was analysed and compared, by DNA hybridization, with that of strains with related O antigens. The possible evolution of the rfb genes in these O antigen groups is discussed.     

The Escherichia coli O111 and Salmonella enterica O35 gene clusters: gene clusters encoding the same colitose-containing O antigen are highly conserved

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. Escherichia coli and Salmonella enterica each have many forms of O antigen, but only three are common to the two species. It has been found that, in general, O-antigen genes are of low GC content. This deviation in GC content from that of typical S. enterica or E. coli genes (51%) is thought to indicate that the O-antigen DNA originated in species other than S. enterica or E. coli and was captured by lateral transfer. The O-antigen structure of Salmonella enterica O35 is identical to that of E. coli O111, commonly found in enteropathogenic E. coli strains. This O antigen, which has been shown to be a virulence factor in E. coli, contains colitose, a 3,6-dideoxyhexose found only rarely in the Enterobacteriaceae. Sequencing of the O35-antigen gene cluster of S. enterica serovar Adelaide revealed the same gene order and flanking genes as in E. coli O111. The divergence between corresponding genes of these two gene clusters at the nucleotide level ranges from 21.8 to 11.7%, within the normal range of divergence between S. enterica and E. coli. We conclude that the ancestor of E. coli and S. enterica had an O antigen identical to the O111 and O35 antigens, respectively, of these species and that the gene cluster encoding it has survived in both species.     

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Characterization of six flagellin genes in the H3, H53 and H54 standard strains of Escherichia coli

Six flagellin genes in three H standard Escherichia coli strains for H3, H53 and H54 were characterized. Each strain has two flagellin genes, one of which is expressed as its standard H antigen. A pair of flagellin genes flkA3 (encoding for H3 antigen) and fliC16 (H16) was cloned from Bi7327-41, flkA53 (H53) and fliC-53 from E480-68, and flmA54 (H54) and fliC-54 from E223-69. Two fliC genes, fliC-53 and fliC-54, are nonfunctional owing to the insertions of IS1 and IS1222, respectively. The flkA and flmA regions are located in the 3' end of the rnpB gene and near the nlpA gene, respectively. Each of them is followed by a gene homologous to fljA, which is known to repress the expression of fliC(i) in Salmonella enterica serovar Typhimurium. These results suggest that they are derived from the same origin of the fljBA operon. However, these regions contain neither the hin gene nor the invertible H segment. The four flagellin genes, fliC16, flkA3, flkA53 and flmA54, share high homology in nucleotide and amino-acid sequences with one another and with the S. enterica serovar Typhimurium flagellin genes. The promoter sequence of fliC16 is homologous to that of fliC(i), whereas the promoter sequences of flkA and flmA are homologous to that of fljB. The terminator sequences of the fliC16, fliC-53 and fliC-54 genes are conserved among themselves and identical with that of the E. coli fliC48 gene. Three FljA repressors, FljA3, FljA53 and FljA54, are homologous highly with one another and moderately with FljA of Salmonella. These results indicate that six flagellin genes analyzed are markedly similar to the Salmonella flagellin genes, suggesting their lateral transfer from Salmonella.     

Salmonella virulence plasmid. Modular acquisition of the spv virulence region by an F-plasmid in Salmonella enterica subspecies I and insertion into the chromosome of subspecies II,IIIa, IV and VII isolates.

The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Molecular diversity of the genetic loci responsible for lipopolysaccharide core oligosaccharide assembly within the genus Salmonella

The waa locus on the chromosome of Salmonella enterica encodes enzymes involved in the assembly of the core oligosaccharide region of the lipopolysaccharide (LPS) molecule. To date, there are two known core structures in Salmonella, represented by serovars Typhimurium (subspecies I) and Arizonae (subspecies IIIA). The waa locus for serovar Typhimurium has been characterized. Here, the corresponding locus from serovar Arizonae is described, and the molecular basis for the distinctive structures is established. Eleven of the 13 open reading frames (ORFs) are shared by the two loci and encode conserved proteins of known function. Two polymorphic regions distinguish the waa loci. One involves the waaK gene, the product of which adds a terminal alpha-1,2-linked N-acetylglucosamine residue that characterizes the serovar Typhimurium core oligosaccharide. There is an extensive internal deletion within waaK of serovar Arizonae. The serovar Arizonae locus contains a novel ORF (waaH) between the waaB and waaP genes. Structural analyses and in vitro glycosyltransferase assays identified WaaH as the UDP-glucose:(glucosyl) LPS alpha-1,2-glucosyltransferase responsible for the addition of the characteristic terminal glucose residue found in serovar Arizonae. Isolates comprising the Salmonella Reference Collections, SARC (representing the eight subspecies of S. enterica) and SARB (representing subspecies I), were examined to assess the distribution of the waa locus polymorphic regions in natural populations. These comparative studies identified additional waa locus polymorphisms, shedding light on the genetic basis for diversity in the LPS core oligosaccharides of Salmonella isolates and identifying potential sources of further novel LPS structures.