A mutational study of the site-specific cleavage of EC83, a multicopy single-stranded DNA (msDNA): nucleotides at the msDNA stem are important for its cleavage.

Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. Such molecules are encoded by genetic elements called retrons. Unlike other retrons, retron EC83 isolated from Escherichia coli 161 produces RNA-free msDNA by site-specific cleavage of msDNA at 5'-TTGA/A-3', where the slash indicates the cleavage site. In order to investigate factors responsible for the msDNA cleavage, retron EC83 was treated with hydroxylamine and colonies were screened for cleavage-negative mutants. We isolated three mutants which were defective in msDNA cleavage and produced RNA-linked msDNA. They were all affected in msd, a gene for msDNA, with a base substitution at the bottom part of the msDNA stem. In contrast, base substitution at and around the cleavage site has no marked effect on msDNA synthesis or its cleavage. From these results, we concluded that the nucleotides at the bottom of the msDNA stem, but not the nucleotides at the cleavage site, play a major role in the recognition and cleavage of msDNA EC83.     

Fructose-Asparagine Is a Primary Nutrient during Growth of Salmonella in the Inflamed Intestine

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10^−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1⁻ SPI2⁻ or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.     

Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.     

Characterization of SEN3800-associated virulence of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis phage type 8

Salmonella is a major public health concern due to the consumption of contaminated food. Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. In this study, transposon mutagenesis identified SEN3800 as a potential binding mutant of SE. Therefore, we hypothesize that SEN3800 plays a role in the colonization ability of SE in the gastrointestinal tract of poultry. To test our hypothesis, we created a mutant of SE in which SEN3800 was deleted. We then tested the in-vitro and in-vivo binding ability of ?SEN3800 when compared to the wild-type and complemented SE strains. Our data showed a significant decrease in the binding ability of ?SEN3800 to T84 intestinal epithelial cells, as well as in the small intestine and cecum of poultry. Furthermore, this binding defect correlated to a defect in invasion, as evidenced by a cell culture model using T84 intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate that SEN3800 contributes to the colonization ability of Salmonella in the gastrointestinal tract of poultry.     

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment,Stem Colonization,and Virulence

Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium''s gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum''s sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.     

Contribution of the Type VI Secretion System Encoded in SPI-19 to Chicken Colonization by Salmonella enterica Serotypes Gallinarum and Enteritidis

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.     

Genome-Wide Screen of Salmonella Genes Expressed during Infection in Pigs, Using In Vivo Expression Technology

Pigs are a food-producing species that readily carry Salmonella but, in the great majority of cases, do not show clinical signs of disease. Little is known about the functions required by Salmonella to be maintained in pigs. We have devised a recombinase-based promoter-trapping strategy to identify genes with elevated expression during pig infection with Salmonella enterica serovar Typhimurium. A total of 55 clones with in vivo-induced promoters were selected from a genomic library of ~10,000 random Salmonella DNA fragments fused to the recombinase cre, and the cloned DNA fragments were analyzed by sequencing. Thirty-one genes encoding proteins involved in bacterial adhesion and colonization (including bcfA, hscA, rffG, and yciR), virulence (metL), heat shock (hscA), and a sensor of a two-component regulator (hydH) were identified. Among the 55 clones, 19 were isolated from both the tonsils and the intestine, while 23 were identified only in the intestine and 13 only in tonsils. High temperature and increased osmolarity were identified as environmental signals that induced in vivo-expressed genes, suggesting possible signals for expression.     

Multicopy single-stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli

Retrons are genetic elements that encode multicopy single-stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.