Molecular phylogenetic typing of pandemic isolates of Salmonella enteritidis

Salmonella enteritidis is now the most common Salmonella serovar in many countries. We have used cloned DNA probes to analyze genome interrelationships between strains chosen to represent the current S. enteritidis pandemic, and included designated type strains of the seven subspecies of Salmonella in order to compare the levels of discrimination of probes. DNA sequence divergence and rearrangements were analyzed in and around the rfa, fim and umuDC loci, and around insertion sites of the Salmonella-specific DNA insertion element, IS200. The S. enteritidis isolates showed a high degree of genome homogeneity. Chromosomal genetic loci exhibited characteristic DNA sequence divergence between subspecies of Salmonella, but no intraserovar divergence or difference with the subspecies I type strain was observed for S. enteritidis. The locus umuDC was not found in S. enteritidis. S. enteritidis contains a conserved and a variable site of insertion of insertion sequence IS200 and the analysis of DNA rearrangements around the second of these sites showed that three distinct evolutionary lines or races exist within pandemic isolates associated with human gasteroenteritis. IS200 profiles of a range of U.K. isolates of the epidemic phage type PT4 showed that all belonged to a single clonal line.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.     

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.     

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe

Abstract A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemology of ovine foot abscess and other diseases caused by F. necrophorum .     

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.     

Identification of Salmonella abortusovis by PCR amplification of a serovar-specific IS200 element.

Field and collection isolates of Salmonella abortusovis carry one IS200 element in a distinct chromosome location. IS200 is not found in the corresponding region of the chromosome of other Salmonella serovars. Sequencing of the boundaries of the S. abortusovis-specific IS200 insertion permitted the design of primers for the amplification of this IS200 element by PCR. Isolates of S. abortusovis are identified by the amplification of a DNA fragment of about 900 bp or larger. PCR amplification of DNA from salmonellae other than S. abortusovis yields either a fragment of about 200 bp or no product. The high specificity of the assay is confirmed by the absence of cross-reactivity with the following templates: (i) sheep DNA, (ii) DNAs from abortion-causing agents other than S. abortusovis, and (iii) DNAs from microorganisms that do not cause abortion but are common in flocks.     

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.     

Tris-dependent site-specific cleavage of Streptomyces lividans DNA

The copy number and location of the insertion sequence IS200, a mobile DNA element, was established across a collection of Salmonella berta. All strains contained one common site, assumed to be present in the evolutionary ancestor of this serovar. With one exception, all strains, including recent outbreak isolates from the UK and sporadic isolates of world-wide distribution, were representatives of a single genotypic clone which carried three common IS200 insertion sites. This clone has acquired diverse combinations of plasmids, reflecting its actual or recent distribution and host. A single isolate, belonging to a second, minor genotypic clone was characterised by two IS200 insertion sites.     

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.     

Molecular features of Mycobacterium avium human isolates carrying a single copy of IS1245 and IS1311 per genome

Human clinical isolates of the Mycobacterium avium complex, from hospitals in Bogotá, were studied using a wide range of molecular tests including PCR restriction-enzyme analysis (PRA) of the hsp65 gene. Up to 21 of the isolates were identified as M. avium PRA variant III (Mav III), a variant obtained only from isolates on the American continent. In contrast to previous reports, restriction fragment length polymorphism analysis using IS1245 and IS1311 showed a single copy for each insertion sequence (IS) in the majority (19/21) of the Colombian Mav III isolates under study. In order to analyse whether the ISs were inserted in a relevant genomic region, experimental conditions were established to determine the insertion loci of each single copy of both ISs in the genome. Analysis of genomic insertion loci indicated that both IS1245 and IS1311 were present in areas containing putatively truncated integrases and/or transposases, which may have an influence on the mobility of the inserted IS. In addition, a conserved genomic region was identified for the insertion of IS1311; this region could be part of the IS1311 itself.     

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of elements.     

Host-mediated modification of PvuII restriction in Mycobacterium tuberculosis.

Restriction endonuclease PvuII plays a central role in restriction fragment length polymorphism analysis of Mycobacterium tuberculosis complex isolates with IS6110 as a genetic marker. We have investigated the basis for an apparent dichotomy in PvuII restriction fragment pattersn observed among strains of the M. tuberculosis complex. The chromosomal regions of two modified PvuII restriction sites, located upstream of the katG gene and downstream of an IS1081 insertion sequence, were studied in more detail. An identical 10-bp DNA sequence (CAGCTGGAGC) containing a PvuII site was found in both regions, and site-directed mutagenesis analysis revealed that this sequence was a target for modification. Strain-specific modification of PvuII sites was identified in DNA from over 80% of the nearly 800 isolates examined. Furthermore, the proportion of modifying and nonmodifying strains differs significantly from country to country.     

Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity

Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain 'fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.     

Bacterial Interspersed Mosaic Elements (Bimes) Are a Major Source of Sequence Polymorphism in Escherichia Coli Intergenic Regions Including Specific Associations with a New Insertion Sequence

A significant fraction of Escherichia coli intergenic DNA sequences is composed of two families of repeated bacterial interspersed mosaic elements (BIME-1 and BIME-2). In this study, we determined the sequence organization of six intergenic regions in 51 E. coli and Shigella natural isolates. Each region contains a BIME in E. coli K-12. We found that multiple sequence variations are located within or near these BIMEs in the different bacteria. Events included excisions of a whole BIME-1, expansion/deletion within a BIME-2 and insertions of non-BIME sequences like the boxC repeat or a new IS element, named IS1397. Remarkably, 14 out of 14 IS1397 integration sites correspond to a BIME sequence, strongly suggesting that this IS element is specifically associated with BIMEs, and thus inserts only in extragenic regions. Unlike BIMEs, IS1397 is not detected in all E. coli isolates. Possible relationships between the presence of this IS element and the evolution of BIMEs are discussed.     

The SHI-3 Iron Transport Island of Shigella boydii 0-1392 Carries the Genes for Aerobactin Synthesis and Transport

In Shigella boydii 0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between lysU and the pheU tRNA gene. DNA sequence analysis of the S. boydii 0-1392 island, designated SHI-3 for Shigella island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the pheU tRNA locus in some S. boydii isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An S. boydii aerobactin synthesis mutant, 0-1392 iucB, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.     

Natural Populations of Escherichia Coli and Salmonella Typhimurium Harbor the Same Classes of Insertion Sequences

Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange. In this study, genetically diverse collections of E. coli and S. typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction. A majority of salmonellae contained IS1 or IS3, elements originally recovered from E. coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E. coli. Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E. coli and S. typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa. This suggests that copies of IS200 have not been recently transferred between E. coli and S. typhimurium and that the element was present in the common ancestor to both species. IS200 is polymorphic within E. coli but homogeneous among isolates of S. typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

IS870 requires a 5'-CTAG-3' target sequence to generate the stop codon for its large ORF1.

The TB regions of the Agrobacterium vitis octopine/cucumopine Ti plasmids constitute a family of related structures. All contain a bacterial insertion element downstream of the TB-iaaM gene, IS870.1. Whereas 43 isolates with octopine/cucumopine Ti plasmids carry only one IS870 copy, strain Ag57 carries a second copy (IS870.2) 3.9 kb to the right of IS870.1 and part of the same TB region. Two other octopine/cucumopine strains carry an IS870 copy on their chromosome (IS870.3). A study of the unmodified insertion sites of IS870.2 and IS870.3, cloned from closely related strains, enabled us to delimit the IS870 elements. IS870 has a size of 1,152 bp and is terminated by inverted repeats. It contains a large open reading frame without a stop codon. However, a stop codon is generated by insertion into the target sequence 5'-CTAG-3'. IS870 is related to five other insertion sequence elements. For two of these, the stop codon of the largest open reading frame is also created by insertion into a CTAG target site.