Diversity of genome structure in Salmonella enterica serovar Typhi populations

The genomes of most strains of Salmonella and Escherichia coli are highly conserved. In contrast, all 136 wild-type strains of Salmonella enterica serovar Typhi analyzed by partial digestion with I-CeuI (an endonuclease which cuts within the rrn operons) and pulsed-field gel electrophoresis and by PCR have rearrangements due to homologous recombination between the rrn operons leading to inversions and translocations. Recombination between rrn operons in culture is known to be equally frequent in S. enterica serovar Typhi and S. enterica serovar Typhimurium; thus, the recombinants in S. enterica serovar Typhi, but not those in S. enterica serovar Typhimurium, are able to survive in nature. However, even in S. enterica serovar Typhi the need for genome balance and the need for gene dosage impose limits on rearrangements. Of 100 strains of genome types 1 to 6, 72 were only 25.5 kb off genome balance (the relative lengths of the replichores during bidirectional replication from oriC to the termination of replication [Ter]), while 28 strains were less balanced (41 kb off balance), indicating that the survival of the best-balanced strains was greater. In addition, the need for appropriate gene dosage apparently selected against rearrangements which moved genes from their accustomed distance from oriC. Although rearrangements involving the seven rrn operons are very common in S. enterica serovar Typhi, other duplicated regions, such as the 25 IS200 elements, are very rarely involved in rearrangements. Large deletions and insertions in the genome are uncommon, except for deletions of Salmonella pathogenicity island 7 (usually 134 kb) from fragment I-CeuI-G and 40-kb insertions, possibly a prophage, in fragment I-CeuI-E. The phage types were determined, and the origins of the phage types appeared to be independent of the origins of the genome types.     

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

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

Gene transfer is a major factor in bacterial evolution

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.      

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.     

Genetic and biochemical characterization of Salmonella enterica serovar typhi deoxyribokinase

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

Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray

The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.     

Waterborne pathogen detection by use of oligonucleotide-based microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

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

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

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening

Salmonella enterica serovar Typhi and Typhimurium are closely related serovars. However, S. Typhi, a human-specific pathogen, has 5% of genes as pseudogenes, far more than S. Typhimurium, which only has 1%. One of these pseudogenes corresponds to sopD2, which in S. Typhimurium encodes an effector protein involved in Salmonella-containing vacuole biogenesis in human epithelial cell lines, which is needed for full virulence of the pathogen. We investigated whether S. Typhi trans-complemented with the functional sopD2 gene from S. Typhimurium (sopD2(STM) ) would reduce the invasion of human epithelial cell lines. Our results showed that the presence of sopD2(STM) in S. Typhi significantly modified the bacterial ability to alter cellular permeability and decrease the CFUs recovered after cell invasion of human epithelial cell line. These results add to mounting evidence that pseudogenes contribute to S. Typhi adaptation to humans.     

Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize

The study was undertaken to understand effects and survival of S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a zoonotic serovar, on maize seed germination and plant growth. All the four strains of S. enterica subspecies enterica serovar Typhimurium significantly reduced germination of maize seeds in sprouting plates as well as in soil. About > or =2.7x10(3) Salmonella cfu ml(-1) of soaking water, while > or =2.7x10(7) Salmonella cfu g(-1) soil were required to significantly inhibit germination of maize. Similar inhibition of germination could be observed using > or = 16 mg of bacteria free Salmonella cell lysate (CL) protein per g of soil or > or =0.5 mg of CL protein per ml of soaking water in sprouting plates. At the constant dose of 3.6x10(7) to 3.8x10(7) Salmonella cfu or 5 mg cell lysate protein ml(-1) of soaking water, four strains of Salmonella significantly reduced germination, however difference between strains was insignificant. After germination too, maize growth was affected both by Salmonella organism and CL with little strain-to-strain variation. All Salmonella persisted in growing plants from 15 to 35 days of plant age and up to 190 days in soil. Maize plants once grown for a week in sterile soil were resistant to invasion of S. enterica subspecies enterica serovar Typhimurium in their leaves even in doses as high as 7.6x10(9) cfu g(-1) of soil. Salmonella persisted better and longer in plants grown from contaminated seed sown in loam soil, but rarely in plants grew in sandy soil. All maize plants had Salmonella in their stumps even after 35 days of sowing irrespective of kind of soil, primary source of infection (soil or seed) and type of S. enterica subspecies enterica serovar Typhimurium strain. The study revealed that Salmonella is not only zoonotic but a phytopathogen also.     

Virulence of 32 Salmonella strains in mice

Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies.     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

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

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

Composition,acquisition, and distribution of the Vi exopolysaccharide-encoding Salmonella enterica pathogenicity island SPI-7

Vi capsular polysaccharide production is encoded by the viaB locus, which has a limited distribution in Salmonella enterica serovars. In S. enterica serovar Typhi, viaB is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA(pheU) sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of viaB-associated DNA in Vi-positive S. enterica serovar Paratyphi C and S. enterica serovar Dublin isolates revealed the presence of similar SPI-7 islands. In S. enterica serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA(pheU) site were absent. In S. enterica serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of S. enterica serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other Salmonella serovars examined, 24 contained no insertions at the equivalent tRNA(pheU) site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA(pheU)-phoN junction, which was not analyzed further. Sequence analysis of the SPI-7 region from S. enterica serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including Pseudomonas aeruginosa and Xanthomonas axonopodis pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.     

Negative osmoregulation of the Salmonella ompS1 porin gene independently of OmpR in an hns background

The ompS1 gene encodes a quiescent porin in Salmonella enterica serovars Typhi and Typhimurium. By using random mariner transposon mutagenesis, mutations that caused derepression of ompS1 expression were isolated, one in S. enterica serovar Typhi and two in S. enterica serovar Typhimurium. All of them mapped in the hns gene in the region coding for the carboxy terminus of the H-NS nucleoid protein. The derepressed ompS1 expression was subject to negative regulation at high osmolarity, both in the presence and in the absence of OmpR. This observation was possible due to the fact that there are two promoters: P1, which is OmpR dependent, and P2, which does not require OmpR for activation (rather, OmpR represses P2). The sequences upstream from position -88, a region previously shown to be involved in the negative regulation of ompS1, can form a static bend, and the integrity of this region was required for function and binding of H-NS and for osmoregulation, as determined with gene reporter fusions of different lengths and with a 31-bp deletion mutant. This is consistent with the notion that this region determines a structure required for repression. Hence, ompS1 shares negative regulation by H-NS with other loci, such as the bgl operon and the ade gene.     

Complete Genome Sequence of Bacteriophage SSU5 Specific for Salmonella enterica serovar Typhimurium Rough Strains

Salmonella enterica serovar Typhimurium rough strain-specific phage SSU5 was isolated, and its whole genome was sequenced. The 103,229-bp-long double-stranded DNA genome of SSU5 encodes 130 open reading frames with one tRNA for asparagine. Genomic analysis revealed that SSU5 might be the phylogenetic origin of cryptic plasmid pHCM2 harbored by Salmonella Typhi CT18.     

2-aminopurine allows interspecies recombination by a reversible inactivation of the Escherichia coli mismatch repair system

2-Aminopurine treatment of Escherichia coli induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a Salmonella enterica serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.