Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E. coli

To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.     

The virulence plasmids of Salmonella.

Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes. Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear. Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system. Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process. The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons. The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.     

A novel transposon-based method for elimination of large bacterial plasmids

Elimination or modification of large plasmids of bacteria is often an essential step in functional analysis of these replicons. However, the conventional plasmid-curing procedures such as ethidium bromide and heat treatment are insufficient in many cases. For instance, curing of the large virulence plasmid of Salmonella Enteritidis 2,102 has failed when these treatments were applied. To overcome the difficulties, a two-step transposon-based curing method has been developed. First, a Tn10-based transposable unit carrying a Km(R) marker gene and the joined IS30 ends transposes from a replication deficient conjugative plasmid into the target replicon. Then, the inducible IS30 transposase, using the highly reactive joined IS30 ends, mediates deletions or gives rise to the loss of the target plasmid. The efficiency of the method has been monitored by the frequency of Km(S) colonies after induction of IS30 transposase, and it was shown that the Km(S) phenotype often accompanied the complete loss of the virulence plasmid or the formation of deletion derivatives. The procedure has been successfully applied also in removing the large virulence plasmid from enterotoxigenic Escherichia coli (ETEC O147), suggesting that the transposon-based method can be a useful tool for eliminating native plasmids in several bacteria.     

Distribution of two Agrobacterium tumefaciens insertion elements in natural isolates: Evidence for stable association between Ti plasmids and their bacterial hosts

Summary A large number of Agrobacterium tumefaciens strains of the biotype III group carry two to ten copies of two related IS elements, IS866 and IS867. A study of the distribution and localization of these elements in 54 strains showed that one IS866 and two IS867 copies are always found at characteristic sites on the octopine/cucumopine and vitopine Ti plasmids, whereas varying amounts of IS866 and IS867 copies occur at different positions on the chromosome. By comparison of the IS patterns, an evolutionary tree could be deduced which shows the phylogenetic relationships between 23 different types of Agrobacterium strains. The structures of the T-regions of the different strains were also compared. Within the octopine/cucumopine group, eight T-region patterns could be defined. These patterns were found to be correlated with the chromosomal IS patterns. This strongly suggests that the IS866 and IS867 containing Ti plasmids are stably associated with their bacterial hosts. The possible role of the IS866 and IS867 elements in Ti plasmid evolution is discussed.     

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.     

Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin. We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.     

Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group

Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12 095 bp), pFR12.5 (12 459 bp) and pFR55 (55 712 bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study of the molecular basis of the conjugative process in Gram positive bacteria, particularly due to the similarity with known conjugation systems. It is also a contribution to the expansion of the non-pathogenic B. cereus plasmid gene pool.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.     

Nucleotide Sequence and Analysis of pBL1, a Bacteriocin-Producing Plasmid from Lactococcus lactis IPLA 972

The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.     

Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868.

Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide. They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB. The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene. Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp. savastanoi. The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868. We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site. The deletion was caused during the second transposition or by later recombination between the two IS868 copies. Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes. Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution.     

Recent horizontal transmission of plasmids between natural populations of Escherichia coli and Salmonella enterica.

Seventy-one natural isolates obtained from a Salmonella reference collection were examined for the presence of plasmids closely related to the Escherichia coli F plasmid. The collection consists of several serovars of the S. enterica Typhimurium complex, subspecies I, to which 99% of pathogenic salmonellae belong. Molecular genetic techniques of DNA hybridization, along with PCR and DNA sequencing, were used to examine the occurrence, distribution, and genetic diversity of F-like plasmids among Salmonella strains. The F plasmid genes examined were finO, traD, traY, and repA, which map at dispersed positions on the F plasmid of E. coli. Comparative sequence analysis of each of the four genes in Salmonella plasmids showed them to be homologous (in some cases, virtually identical) to those found in F plasmids of E. coli natural isolates. Furthermore, the frequency of F-like plasmids in Salmonella strains was approximately the same as that observed in the E. coli Reference Collection. However, in Salmonella, the distribution was confined predominately to the serovars Typhimurium and Muenchen. The unexpected finding of a shared pool of F-like plasmids between S. enterica and E. coli demonstrates the significant role of conjugation in the histories of these important bacterial species.     

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.     

Evolution of the virulence plasmids of non-typhoid Salmonella and its association with antimicrobial resistance

Among more than 2,500 serovars, eight contain a virulence plasmid, including medically important Salmonella enterica serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium. These serovar-specific virulence plasmids vary in size, but all contain the spv operon, which plays a role in the expression of the virulence. Genetically, these virulence plasmids are likely derived from a common ancestral plasmid possessing virulence-related genes and loci. Based on the analysis of the available DNA sequences of the plasmids, the phylogenetic path may be split into two: pSPV (virulence plasmid of S. Gallinarum-Pullorum) acquires an incompatibility-related locus that differs from that of the others. At some point, pSCV (virulence plasmid of S. Choleraesuis) and pSDV (virulence plasmid of S. Dublin) lose oriT by recombination or simply by deletion, making the two unable to be mobilized. On the other hand, pSEV (virulence plasmid of S. Enteritidis) also loses some DNA by deletion but not as extensively as pSCV, and therefore pSEV is closest to pSTV (virulence plasmid of S. Typhimurium) both genetically and biologically. The pSTV shows the least alternation during the evolution. There are two types of pSDV. pSDVu recombines with non-virulence 36.6-kb plasmid to acquire additional incompatibility trait to form pSDVr. Recent reports indicated that S. Choleraesuis and S. Typhimurium could generate different types of hybrid plasmids, which consisted of the serovar-specific virulence plasmid and an array of resistance gene cassettes. The recombination gives Salmonella a survival advantage in an unfavorable drug environment. The integration of resistance genes and additional replicons into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and poses a serious threat to public health.     

Diagnostic and epidemiological significance of F-donor activity of Enterobacteria strains

The study of the circulating strains of enteropathogenic Escherichia coli (EPEC), shigellae and salmonellae for the presence of F-factor (the conjunction factor) was carried out. The study revealed that 85% of Shigella flexneri strains 2a had conjugative (F)-factor. The proportion of such strains was 63% among EPEC of different serovars and 15% among Salmonella enteritidis. As 63% EPEC strains had hemolytic activity and were found to carry F-conjugative plasmid it should be taken into consideration in the identification of microorganisms. The presence of conjugation plasmids is an important epidemiological marker.     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.