DNA amplification and rearrangements in archival Salmonella enterica serovar Typhimurium LT2 cultures

Variations in genome size and gene order were observed in archival Salmonella enterica serovar Typhimurium cultures stored for over 40 years. In one strain, microarray analysis revealed a large, stable amplification. PCR analysis of the same strain revealed a genomic duplication that underwent a translocation. Other strains had smaller duplications and deletions. These results demonstrate that storage in stabs over time at room temperature not only allows for further bacterial growth but also may produce an environment that selects for a variety of mutations, including genomic rearrangements.     

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.     

Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and nonprophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization

Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.     

Virulence plasmid interchange between strains ATCC 14028, LT2, and SL1344 of Salmonella enterica serovar Typhimurium

Strains ATCC 14028 and SL1344 of Salmonella enterica serovar Typhimurium are more virulent than LT2 in the BALB/c mouse model. Virulence plasmid swapping between strains ATCC 14208, LT2, and SL1344 does not alter their competitive indexes during mouse infection, indicating that the three plasmids are functionally equivalent, and that their contribution to virulence is independent from the host background. Strains ATCC 14028 and LT2 are more efficient than SL1344 as conjugal donors of the virulence plasmid. Virulence plasmid swapping indicates that reduced ability of conjugal transfer is a property of the SL1344 plasmid, not of the host strain. An A→V amino acid substitution in the TraG protein appears to be the major cause that reduces conjugal transfer in the virulence plasmid of SL1344. Additional sequence differences in the tra operon are found between the SL1344 plasmid and the ATCC 14028 and LT2 plasmids. Divergence in the tra operon may reflect the occurrence of genetic drift either after laboratory domestication or in the environment. The latter might provide evidence that possession of conjugal transfer functions is a neutral trait in Salmonella populations, a view consistent with the abundance of Salmonella isolates whose virulence plasmids are non-conjugative.     

Two DNA invertases contribute to flagellar phase variation in Salmonella enterica serovar Typhimurium strain LT2

Salmonella enterica serovar Typhimurium strain LT2 possesses two nonallelic structural genes, fliC and fljB, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the fljB promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene fin. We constructed hin and fin disruption mutants from LT2 and examined their phase variation abilities. The hin disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the fin disruption mutant could undergo phase variation, fin hin double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring hin mutation, lacks Fels-2, which ensures the phase stability in this serovar.     

Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.     

Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences

The type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oligonucleotide external guide sequences (EGSs) complementary to invB or invC mRNA. These EGSs direct single site cleavage in these mRNAs by endogenous RNase P, and their expression in Salmonella results in invC mRNA and InvC protein depletion, decreased type III secretion and interference with host cell invasion. Comparison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.     

Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis

The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned allele numbers, which were used for strain comparison.     

Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.     

Biofilm formation by Salmonella enterica serovar Typhimurium colonizing solid tumours

Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella-colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.     

Direct and quantitative analysis of Salmonella enterica serovar Typhimurium using real-time PCR from artificially contaminated chicken meat

For quantitative PCR assay of Salmonella enterica serovar Typhimurium in food samples, a real-time PCR method was developed, based on DNA genome equivalent. Specific primers and probe designed based on the STM4497 gene of S. Typhimurium LT2 showed the specificity to S. Typhimurium. Threshold cycle (Ct) values of real-time PCR were obtained from a quantitative standard curve with genomic DNA of Salmonella Typhimurium. In addition, the recovery of S. Typhimurium inoculated artificially to chicken samples with 4.5x105 to 4.5x100 CFU/ml was evaluated by using real-time PCR and plate-count methods. Result showed that the number of cells calculated from the real-time PCR method had good correlation with that of the plate-count method. This real-time PCR method could be applicable to the detection and quantification of S. Typhimurium in food samples.     

Hydrogen-stimulated carbon acquisition and conservation in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium can utilize molecular hydrogen for growth and amino acid transport during anaerobic growth. Via microarray we identified H(2) gas-affected gene expression changes in Salmonella. The addition of H(2) caused altered expression of 597 genes, of which 176 genes were upregulated and 421 were downregulated. The significantly H(2)-upregulated genes include those that encode proteins involved in the transport of iron, manganese, amino acids, nucleosides, and sugars. Genes encoding isocitrate lyase (aceA) and malate synthase (aceB), both involved in the carbon conserving glyoxylate pathway, and genes encoding the enzymes of the d-glucarate and d-glycerate pathways (gudT, gudD, garR, garL, garK) are significantly upregulated by H(2). Cells grown with H(2) showed markedly increased AceA enzyme activity compared to cells without H(2). Mutant strains with deletion of either aceA or aceB had reduced H(2)-dependent growth rates. Genes encoding the glutamine-specific transporters (glnH, glnP, glnQ) were upregulated by H(2), and cells grown with H(2) showed increased [(14)C]glutamine uptake. Similarly, the mannose uptake system genes (manX, manY) were upregulated by H(2,) and cells grown with H(2) showed about 2.0-fold-increased [(14)C]d-mannose uptake compared to the cells grown without H(2). Hydrogen stimulates the expression of genes involved in nutrient and carbon acquisition and carbon-conserving pathways, linking carbon and energy metabolism to sustain H(2)-dependent growth.     

Identification of host-specific colonization factors of Salmonella enterica serovar Typhimurium

The severity of infections caused by Salmonella enterica serovar Typhimurium varies depending on the host species. Numerous virulence genes have been identified in S. Typhimurium, largely from studies in mice, but their roles in infections of other species remain unclear. In the most comprehensive survey of its kind, through the use of signature-tagged mutagenesis of S. Typhimurium we have identified mutants that were unable to colonize calf intestines, mutants unable to colonize chick intestines and mutants unable to colonize both species. The type three secretion systems encoded on Salmonella pathogenicity islands (SPIs) 1 and 2 were required for efficient colonization of cattle. However, disruption of these secretion systems only caused a minor defect in S. Typhimurium colonization of chicks. Transposon insertions in SPI-4 compromised S. Typhimurium colonization of cattle, but not chicks. This is the first data confirming a role for SPI-4 in pathogenesis. We have also been able to ascribe a role in colonization for cell surface polysaccharides, cell envelope proteins, and many 'housekeeping' genes and genes of unknown function. We conclude that S. Typhimurium uses different strategies to colonize calves and chicks. This has major implications for vaccine design.