RESUMO
Salmonella enterica subspecies I, serovar Typhimurium (S. typhimurium), is a leading cause of human gastroenteritis, and is used as a mouse model of human typhoid fever. The incidence of non-typhoid salmonellosis is increasing worldwide, causing millions of infections and many deaths in the human population each year. Here we sequenced the 4,857-kilobase (kb) chromosome and 94-kb virulence plasmid of S. typhimurium strain LT2. The distribution of close homologues of S. typhimurium LT2 genes in eight related enterobacteria was determined using previously completed genomes of three related bacteria, sample sequencing of both S. enterica serovar Paratyphi A (S. paratyphi A) and Klebsiella pneumoniae, and hybridization of three unsequenced genomes to a microarray of S. typhimurium LT2 genes. Lateral transfer of genes is frequent, with 11% of the S. typhimurium LT2 genes missing from S. enterica serovar Typhi (S. typhi), and 29% missing from Escherichia coli K12. The 352 gene homologues of S. typhimurium LT2 confined to subspecies I of S. enterica-containing most mammalian and bird pathogens-are useful for studies of epidemiology, host specificity and pathogenesis. Most of these homologues were previously unknown, and 50 may be exported to the periplasm or outer membrane, rendering them accessible as therapeutic or vaccine targets.
Assuntos
Genoma Bacteriano , Salmonella typhimurium/genética , Animais , Cromossomos Bacterianos , DNA Bacteriano , Enterobacteriaceae/genética , Escherichia coli/genética , Fímbrias Bacterianas/genética , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Pseudogenes , Vacinas contra Salmonella/genética , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Análise de Sequência de DNA , Especificidade da Espécie , Virulência/genéticaRESUMO
Intervening sequences (IVSs) in the rrl genes for 23S rRNA are transcribed but later removed by RNase III without religation during RNA processing, leading to fragmented rRNA. We examined about 240 strains of the family Enterobacteriaceae for presence of IVSs using PCR. No IVSs were detected in strains belonging to Escherichia, Shigella, Enterobacter, Erwinia, Ewingella, Hafnia, Kluyvera, Morganella, Pantoea, or Serratia. Previously unreported IVSs were detected in Klebsiella oxytoca, Citrobacter amalonaticus, and Providencia stuartii; previously reported IVSs are in species of Salmonella, Proteus, Providencia, and Yersinia. The sporadic distribution of IVSs indicates lateral genetic transfer of IVSs.
Assuntos
Genes de RNAr , Íntrons , RNA Ribossômico 23S/genética , Sequência de Bases , Enterobacteriaceae/genética , Genes Bacterianos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , Alinhamento de SequênciaRESUMO
Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.
Assuntos
DNA Ribossômico/genética , RNA Ribossômico 23S/genética , Salmonella/genética , Sequência de Bases , DNA Ribossômico/química , Variação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Ribossômico 23S/química , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Intervening sequences (IVSs) occur sporadically in several bacterial genera in the genes for 23S rRNA at relatively conserved locations. They are cleaved after transcription and lead to the presence of fragmented rRNA, which is incorporated into the ribosomes without religation but is nevertheless functional. The fragmentation of rRNA and the number of IVSs in all 72 strains of the Salmonella Reference Collection B set and 16 strains of the Salmonella Reference Collection C set, which have been established on the basis of multilocus enzyme electrophoresis (MLEE), were analyzed in the present study. Fragmentation of 23S rRNA was restricted to conserved cleavage sites located at bp 550 (helix 25) and bp 1170 (helix 45), locations where IVSs have been reported. Random cleavage at sites where IVSs could not be detected was not seen. Uncleaved IVSs were not detected in any case; thus, the IVSs invariably led to rRNA fragmentation, indicating a strong selection for maintenance of RNase III cleavage sites. The distribution of the number of IVSs carried by the different strains in the seven rrl genes is diverse, and the pattern of IVS possession could not be related to the MLEE pattern among the various Salmonella strains tested; this indicates that the IVSs are frequently exchanged between strains by lateral transfer. All eight subspecies of the genus Salmonella, including subspecies V represented by Salmonella bongori, have IVSs in both helix 25 and helix 45; this indicates that IVSs entered the genus after its divergence from Escherichia coli (more than 100 million years ago) but before separation of the genus Salmonella into many forms or that they were in the ancestor but have been lost from Escherichia.
Assuntos
Proteínas de Escherichia coli , Filogenia , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico 23S/genética , Sequências Reguladoras de Ácido Nucleico/genética , Salmonella/classificação , Salmonella/genética , Pareamento de Bases/genética , Bancos de Espécimes Biológicos , Sequência Conservada/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Genes Bacterianos/genética , Variação Genética/genética , Modelos Genéticos , Peso Molecular , Reação em Cadeia da Polimerase , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Recombinação Genética/genética , Sequências Reguladoras de Ácido Nucleico/fisiologia , Ribonuclease III , Óperon de RNAr/genéticaRESUMO
Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.
Assuntos
Genes de RNAr/genética , Íntrons/genética , Proteus/genética , Providencia/genética , RNA Ribossômico 23S/genética , Sequência de Bases , Northern Blotting , Eletroforese em Gel de Campo Pulsado , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Proteus/metabolismo , Providencia/metabolismo , RNA Ribossômico 23S/metabolismo , Análise de Sequência de DNARESUMO
Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.
Assuntos
Genoma Bacteriano , Pasteurella/genética , Filogenia , Salmonella/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Eletroforese em Gel de Campo Pulsado , Endodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Pasteurella/classificação , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Salmonella/classificaçãoRESUMO
Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e. g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.
Assuntos
Genes de RNAr/genética , Recombinação Genética , Salmonella typhi/classificação , Salmonella typhi/genética , Técnicas de Tipagem Bacteriana , Southern Blotting , Sondas de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos/genética , Genoma Bacteriano , Genótipo , Óperon de RNArRESUMO
Partial digestion with I-CeuI, which digests bacterial DNA at the gene coding for the large subunit rRNA, established the rrn genomic skeleton (the distance in kb between rRNA operons) in 56 strains of Salmonella, from Salmonella Reference B (SARB) set. All had seven I-CeuI sites, indicating seven rrn operons. The order of I-CeuI fragments was ABCDEFG in S. typhimurium LT2 and in 31 other species, mostly host-generalists; in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum (host-specialized species), these fragments are rearranged, due to homologous recombination between the rrn operons. Rearrangements, such as inversions and translocations not involving the rrn operons, are rare. I-CeuI fragments of some species are larger than the norm, suggesting the insertion of unique blocks of DNA by lateral transfer from other species.
Assuntos
Cromossomos Bacterianos/genética , Recombinação Genética , Salmonella/genética , Óperon de RNAr/genética , DNA Bacteriano , Eletroforese em Gel de Campo Pulsado , Endodesoxirribonucleases/metabolismo , RNA Ribossômico/genética , Mapeamento por Restrição , Especificidade da EspécieRESUMO
Early genetic studies showed conservation of gene order in the enteric bacteria. Two recent methods using pulsed field gel electrophoresis (PFGE) to determine the physical map of the genome are: (i) partial digestion with the endonuclease I-CeuI, which digests the DNA of bacteria in the rrn operon for rRNA (ribosomal RNA), thus establishing the "rrn genomic skeleton" (the size in kbp of the intervals between rRNA operons); (ii) analysis of XbaI and B1nI sites within Tn10 insertions in the chromosome. The order of I-CeuI fragments, which is ABCDEFG in S. typhimurium LT2 and E. coli K-12, was found to be conserved in most Salmonella species, most of which grow in many hosts (host-generalists). However, in S. typhi, S. paratyphi C, S. gallinarum, and S. pullorum, species which are host-specialized, these fragments are rearranged, due to homologous recombination between the rrn operons, resulting in translocations and inversions. Inversions and translocations not involving the rrn operons are seldom detected except for inversions over the TER (termination of replication) region. Additive genetic changes (due to lateral transfer resulting in insertion of nonhomologous DNA) have resulted in "loops" containing blocks of DNA which provide new genes to specific strains, thus driving rapid evolution of new traits.
Assuntos
Cromossomos Bacterianos , Escherichia coli/genética , Recombinação Genética , Salmonella/genética , Rearranjo GênicoRESUMO
Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90-110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation. In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S. typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12. The resulting strain of S. typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E. coli. These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes.
Assuntos
Exorribonucleases/deficiência , RNA Bacteriano/química , RNA Ribossômico 23S/química , Salmonella typhimurium/genética , Sequência de Bases , Elementos de DNA TransponíveisRESUMO
Gene order in the chromosomes of Escherichia coli K-12 and Salmonella typhimurium LT2, and in many other species of Salmonella, is strongly conserved, even though the genera diverged about 160 million years ago. However, partial digestion of chromosomal DNA of Salmonella typhi, the causal organism of typhoid fever, with the endonuclease I-CeuI followed by separation of the DNA fragments by pulsed-field gel electrophoresis showed that the chromosomes of independent wild-type isolates of S. typhi are rearranged due to homologous recombination between the seven rrn genes that code for ribosomal RNA. The order of genes within the I-CeuI fragments is largely conserved, but the order of the fragments on the chromosome is rearranged. Twenty-one different orders of the I-CeuI fragments were detected among the 127 wild-type strains we examined. Duplications and deletions were not found, but transpositions and inversions were common. Transpositions of I-CeuI fragments into sites that do not change their distance from the origin of replication (oriC) are frequently detected among the wild-type strains, but transpositions that move the fragments much further from oriC were rare. This supports the gene dosage hypothesis that genes at different distances from oriC have different gene dosages and, hence, different gene expression, and that during evolution genes become adapted to their specific location; thus, cells with changes in gene location due to transpositions may be less fit. Therefore, gene dosage may be one of the forces that conserves gene order, although its effects seem less strong in S. typhi than in other enteric bacteria. However, both the gene dosage and the genomic balance hypotheses, the latter of which states that the origin (oriC) and terminus (TER) of replication must be separated by 180 degrees C, need further investigation.
Assuntos
Cromossomos Bacterianos , Salmonella typhi/genética , Evolução Biológica , Mapeamento Cromossômico , DNA Ribossômico/genética , Enterobacteriaceae/genética , Genes Bacterianos , Íntrons , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Recombinação Genética , Salmonella typhi/crescimento & desenvolvimento , Transcrição GênicaRESUMO
Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.
Assuntos
Íntrons , RNA Ribossômico 23S/genética , Salmonella typhi/genética , Sequência de Bases , Evolução Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA BacterianoRESUMO
The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.
Assuntos
DNA Ribossômico , Genes Bacterianos , Íntrons , RNA Ribossômico 23S/genética , Salmonella typhimurium/genética , Sequência de Bases , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
Four fimbrin-encoding genes, fimA (type-1 or SEF21 fimbriae), agfA (thin aggregative or SEF17 fimbriae), sefA (SEF14 fimbriae and sefD (SEF18 fimbriae) from Salmonella enteritidis (Se) 27655-3b were located onto the XbaI-BlnI genomic restriction maps of Salmonella typhimurium (St) LT2 and Se strains SSU7998 and 27655-3b. The XbaI or BlnI genomic fragments carrying these genes were identified by hybridization with labeled oligodeoxyribonucleotides or fimbrin-encoding genes. The fimbrin-encoding genes were not encoded by the virulence plasmids, but were located on chromosomal DNA fragments. The position of each gene on a given XbaI fragment was determined by hybridization of a series of XbaI-digested genomic DNA samples from previously characterized Tn10 mutants of Se and St with its respective probe. The fimA gene mapped near 13 centisomes (Cs) between purE884::Tn10 at 12.6 Cs (11.8 min) and apeE2::Tn10 at 12.8 Cs (12.3 min) beside the first XbaI site at 13.0 Cs in St or between purE884::Tn10 at 12.6 Cs and the XbaI site at 13.6 Cs in Se. The agfA gene mapped near 26 Cs between putA::Tn10 and pyrC691::Tn10 in St, but near 40 Cs between pncX::Tn10 and the XbaI site at 43.3 Cs in Se. This difference in map position was due to the location of agfA near one end of the 815-kb chromosomal fragment inverted between Se and St. The sefA and sefD genes mapped precisely at 97.6 Cs in Se, but were absent from the genome of St LT2. To verify the mapping procedures used herein, tctC was also mapped in both Salmonella serovars. As expected, tctC mapped near 60 Cs in both St and Se, thereby confirming previous studies.
Assuntos
Antígenos de Bactérias , Proteínas de Fímbrias , Fímbrias Bacterianas/genética , Genes Bacterianos , Salmonella enteritidis/genética , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Sequência de Bases , Moléculas de Adesão Celular/genética , Mapeamento Cromossômico , DNA Bacteriano/genética , Mapeamento por RestriçãoRESUMO
Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.
Assuntos
Inversão Cromossômica , Cromossomos Bacterianos , RNA Ribossômico/genética , Recombinação Genética , Salmonella paratyphi A/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , Eletroforese em Gel de Campo Pulsado , Endodesoxirribonucleases , Rearranjo Gênico , Peso Molecular , Mapeamento por Restrição , Salmonella typhimurium/genéticaRESUMO
The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis. The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively. The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, and labelling with 32P, and reelectrophoresis and named in alphabetical order. Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S. typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10. All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S. typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S. typhimurium LT2 to AGCEFDB in S. typhi. In addition, there is a 500-kb inversion which covers the terminus region. Comparisons of lengths of segments between genes showed that S. typhi has segments which differ in size from those in S. typhimurium. The viaB locus, for synthesis of the Vi antigen of S. typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.
Assuntos
Genoma Bacteriano , Polissacarídeos Bacterianos , Mapeamento por Restrição , Salmonella typhi/genética , Salmonella/genética , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Inversão Cromossômica , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/metabolismo , Genes Bacterianos/genética , Salmonella typhi/classificação , Alinhamento de SequênciaRESUMO
We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.
Assuntos
Mapeamento Cromossômico , Genes Bacterianos/genética , Salmonella typhimurium/genéticaRESUMO
The enzyme I-CeuI, encoded by a class I mobile intron inserted in the gene for 23S rRNA in Chlamydomonas eugamatos, cleaves a specific 19-bp sequence in this gene. This sequence is present only in the seven genes for rRNA in Salmonella typhimurium and Escherichia coli. Partial digestion with I-CeuI of DNA from 17 wild-type strains of S. typhimurium indicates that the chromosome of these strains is strongly conserved, for the digestion products closely resemble those of strain LT2. The lengths and order of chromosomal segments are conserved in 15 of the strains; 2 show some rearrangements. XbaI digestion indicated heterogeneity without revealing the genomic structure. Because of conservation of I-CeuI sites in genes for rRNA and conservation of the number and locations of these genes, I-CeuI provides an excellent tool for the rapid examination of the chromosomes of related species of bacteria; differences in the fingerprints indicate the occurrence of chromosomal rearrangements such as insertions or inversions.
Assuntos
Endodesoxirribonucleases/metabolismo , Salmonella typhimurium/genética , Animais , Chlamydomonas/enzimologia , DNA Bacteriano/química , DNA Ribossômico/genética , Eletroforese em Gel de Campo Pulsado , Mapeamento por Restrição , Especificidade da EspécieRESUMO
We have determined the genomic map of the bacterium Salmonella typhi Ty2, the causal organism of typhoid fever, by using pulsed-field gel electrophoresis. Digestion of the Ty2 genome with endonucleases Xba I, Bln I, and Ceu I yielded 33, 26, and 7 fragments, respectively, that were placed in order on a circular chromosome of 4780 kb. Transposon Tn10 was inserted in specific genes of Salmonella typhimurium and transduced into S. typhi, and thus, the positions of 37 S. typhi genes were located through the Xba I and Bln I sites of the Tn10. Gene order on chromosomes of Escherichia coli K-12 and S. typhimurium LT2 is remarkably conserved; however, the gene order in S. typhi Ty2 is different, suggesting it has undergone major genomic rearrangements during its evolution. These rearrangements include inversions and transpositions in the 7 DNA fragments between the seven rrn operons for rRNA (postulated to be due to homologous recombination in these rrn genes), another inversion that covers the replication terminus region (resembling inversions found in other enteric bacteria), and at least three insertions, one as large as 118 kb. Partial digestion of genomic DNA with the intron-encoded endonuclease I-Ceu I, which cuts only in rrn genes, shows chromosomal rearrangements, apparently due to homologous recombination in the rrn genes, that were detected in all wild-type strains of S. typhi tested. These rearrangements may have been selected to compensate for the insertions that otherwise would have altered the locations of genes with respect to the origin and terminus of replication. These observations are relevant to our view of the evolution of the bacterial genome and may be significant in the virulence of S. typhi.