Role of trehalose in growth at high temperature of Salmonella enterica serovar Typhimurium

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of Salmonella enterica serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45 degrees C. One of these mutations proved to be in the seqA gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with pgm (which encodes phosphoglucomutase), it is likely to be polar on the expression of the pgm gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the seqA::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an otsA mutation, which inactivates the first step of the trehalose biosynthetic pathway. The otsA mutation caused a growth defect at 45 degrees C in minimal medium containing 0.2 M NaCl that was similar to that caused by the pgm mutation, but otsA did not affect growth rate in this medium at 37 degrees C. These results suggest that the growth defect of the seqA-pgm mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45 degrees C had a fivefold higher trehalose pool size than cells grown at 30 degrees C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.     

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.     

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.     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.     

Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.     

Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S. Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

Autophagy Recognizes Intracellular Salmonella enterica serovar Typhimurium in Damaged Vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S.Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

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.