Evolutionary lines among Salmonella enteritidis phage types are identified by insertion sequence IS200 distribution

A survey was made of the presence, copy number and location of the Salmonella-specific DNA insertion element IS200, within the genomes of the 27 phage type strains of Salmonella enteritidis. All the phage type strains contained copies of IS200 revealed by genomic Southern blot hybridizations with a 300-bp DNA probe internal to the element. Restriction site variation around IS200 insertion sites was examined. Three fundamental patterns of hybridization corresponding to chromosomal IS200 loci were found. In terms of population genetics, these 'IS200 profiles' correspond to clonal lineages of recent evolutionary origin, and underline the phage-typing scheme for epidemiological subdivision of S. enteritidis. The molecular analysis is consistent with genetic selection pressures which are apparent in the observed epidemiological distribution of S. enteritidis, since each clonal lineage contained one of the phage types of major clinical importance in the U.K.     

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.     

Characterization of Salmonella virchow phage types by plasmid profile and IS200 distribution

The type strains of the 57 phage types of Salmonella virchow have been characterized by plasmid profile and by distribution of the insertion sequence IS 200 . Thirty-two strains carried plasmids and 21 profile types were identified; 17 strains were resistant to antimicrobial agents. In contrast only six of the type strains carried IS 200 elements and three patterns were identified. Within Salm. virchow phage type 31, five of 10 wild-type isolates carried plasmids and two plasmid profiles were identified; in contrast, an IS 200 element was identified in the genome of only one of these strains. It is concluded that for Salm. virchow , IS 200 is unlikely to significantly extend the degree of discrimination achieved by phage typing which may be supplemented when appropriate by plasmid profile typing.     

Phage type 193 of Salmonella typhimurium contains different chromosomal genotypes and multiple IS 200 profiles

Abstract The common phage type 193 of Salmonella typhimurium was analyzed with respect to molecular markers of chromosomal genotype. Three profiles of the 16S rRNA genes and seven profiles of the DNA insertion element of IS2 00 were found among tne representative strains of DT193. The IS 200 profiles found within this single phage type were highly diverse, confirming that DT193 is a composite phage type containing several distinct clones and hybrid lines. IS2 00 profiling is thus appropriate both for primary strain discrimination, and for subdivision within certain phage types of S. typhimurium , such as DT193. This rapid molecular definition of clonality will be useful for the epidemiological investigation of food poisoning outbreaks.     

转座子随机插入鉴定肠炎沙门氏菌生物膜形成相关基因

[目的]生物膜在沙门氏菌的致病性和引起沙门氏菌食物中毒等方面起着重要作用,本研究为了鉴定影响沙门氏菌生物膜形成的基因.[方法]利用结晶紫染色定量法对74株鸡源的肠炎、鸡白痢和鸡伤寒沙门氏菌进行生物膜测定,选择生物膜生长较好的肠炎沙门氏菌C050041,采用转座子随机插入法构建突变株库.[结果]84%的鸡源沙门氏菌菌株可在塑料表面形成生物膜;通过转座子插入获得1924个突变株,筛选的生物膜降低突变株经生长曲线测定、测序和序列比对及Southern blot分析鉴定出15个插入基因,它们分别为metE、ompR、rpoS、,和G、rfaJ、rfaK、rfaP、rfbH、rhlE、spiA、steB、tpx、ybdN和2个未知功能的基因.[结论]我们鉴定出了多个影响生物膜形成的新基因,这些基因的发现为进一步研究沙门氏菌生物膜形成的调控机制,研制减毒沙门氏菌疫苗奠定了基础.     

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.     

Sequence analysis and distribution of an IS3-like insertion element isolated from Salmonella enteritidis

The nucleotide sequence of a 3 kb region immediately upstream of the sef operon of Salmonella enteritidis was determined. A 1230 base pair insertion sequence which shared sequence identity (>75%) with members of the IS3 family was revealed. This element, designated IS1230, had almost identical (90% identity) terminal inverted repeats to Escherichia coli IS3 but unlike other IS3-like sequences lacked the two characteristic open reading frames which encode the putative transposase. S. enteritidis possessed only one copy of this insertion sequence although Southern hybridisation analysis of restriction digests of genomic DNA revealed another fragment located in a region different from the sef operon which hybridised weakly which suggested the presence of an IS1230 homologue. The distribution of IS1230 and IS1230-like elements was shown to be widespread amongst salmonellas and the patterns of restriction fragments which hybridised differed significantly between Salmonella serotypes and it is suggested that IS1230 has potential for development as a differential diagnostic tool.     

Lack of hotspot targets: a constraint for IS30 transposition in Salmonella

IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

Strain typing with IS200 fingerprints in Salmonella abortusovis.

A collection of Salmonella abortusovis isolates was examined for the presence of insertion element IS200. All proved to contain three or four copies of the element. One IS200 hybridization band of approximately 9 kb was found in all isolates, indicating that all S. abortusovis strains carry an IS200 element in similar or identical locations; this band can be potentially useful for serovar identification. S. abortusovis collection isolates from distinct geographic areas were highly polymorphic, suggesting that IS200 fingerprints might provide information on the geographic origin of S. abortusovis strains. Isolates obtained from the same geographic area (the island of Sardinia, Italy) were less polymorphic: all shared three constant IS200 hybridization bands, indicating that they derive from a single ancestor. Most strains analyzed contained an additional copy of IS200 in the variable region of the virulence plasmid. Certain Sardinian flocks proved to be infected by only one S. abortusovis strain, while others harbored two strains. Strain typing with IS200 fingerprints proved to be more reliable than plasmid analysis, because the latter yielded a high degree of polymorphism, even among isolates from the same flock.     

Rapid classification and identification of salmonellae at the species and subspecies levels by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.     

Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12

An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.      

Conventional and molecular approaches to isolates of Salmonella hadar from sporadic and epidemic cases

In September 1994 an outbreak of gastroenteritis occurred in 437 people who had consumed lunch in the canteen of a factory in Central Italy. Salmonella sp. was isolated from stools of 99 patients and in 73 of them Salmonella hadar was identified. This is the first outbreak caused by this serotype described in Italy. In order to examine the genotypic basis of the epidemic strains, molecular typing was applied to sporadic strains isolated before and after the outbreak episode. For this purpose phage type, resistance to antibiotics, DNA plasmid profile and sites of insertion of the mobile element of IS200 were determined. The epidemic strains were genetically distinct from the non-epidemic isolates; they were shown to be phage type 26, harbouring four small plasmids, were resistant to nalidixic acid and showed a unique characteristic IS200 fingerprint. The typing methods used in this study allowed the identification and discrimination of the outbreak strains from related isolates. They can thus be considered as a tool for epidemiological purposes. In addition we should point out the emerging resistance to nalidixic acid, largely used in veterinary medicine, in Salm . hadar .     

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.     

Detection of peptidoglycan and β-glucan with silkworm larvae plasma test

Abstract Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.     

The insertion sequence IS200 fingerprints chromosomal genotypes and epidemiological relationships in Salmonella heidelberg.

In Salmonella heidelberg the copy number of the Salmonella-specific insertion element IS200 was found to vary from four to six. All strains tested contained at least one common insertion site which was serovar specific, and most strains contained three common sites. Concurrent analysis of plasmids indicated that all insertion sequence copies were chromosomally located, and also supported the equivalence of an IS200 fingerprint and clonality. Seven intra-serovar clonal lines were thereby identified. One of these was associated with human infections, including septicaemias. Another was associated with chicken as a host: all these strains also carried a unique plasmid of 23 MDa, which was typed as a member of the IncX group. The chromosomal fingerprint of a third clone showed it to be a descendant of the chicken line marked by a single IS200 transposition. One or two representatives of four other clonal lines were identified. These lines of S. heidelberg could be related by divergent evolution, and the most recent relatives conformed to a continuous branching process model of IS200 transposition. This insertion sequence provided a highly discriminatory molecular marker of the S. heidelberg chromosome, and two of the seven clonal lines so identified were associated with distinct clinical/epidemiological contexts.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.