Lack of YggX results in chronic oxidative stress and uncovers subtle defects in Fe-S cluster metabolism in Salmonella enterica

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking yggX have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between isc mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci, gshA, apbC, apbE, and rseC. The latter mutants share similarities with isc mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from isc mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of isc mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

SigE is a chaperone for the Salmonella enterica serovar Typhimurium invasion protein SigD

SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His(6) on a nickel column.     

Salmonella enterica serovar Typhimurium DT104 displays a rugose phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25 degrees C after 3 days of incubation. Incubation at 37 degrees C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25 degrees C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.     

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.     

Bile-induced curing of the virulence plasmid in Salmonella enterica serovar Typhimurium

Exposure to bile induces curing of the virulence plasmid in Salmonella enterica serovar Typhimurium (pSLT). Disruption of the ccdB gene increases pSLT curing, both spontaneous and induced by bile, suggesting that the pSLT ccdAB genes may encode a homolog of the CcdAB addiction module previously described in the F sex factor. Unlike the F sex factor, synthesis of pSLT-encoded pili does not confer bile sensitivity. These observations may provide insights into the evolution of virulence plasmids in Salmonella subspecies I, as well as the causes of virulence plasmid loss in other Salmonella subspecies.     

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.     

Salmonella enterica strains lacking the frataxin homolog CyaY show defects in Fe-S cluster metabolism in vivo

In Salmonella enterica, the isc operon contains genes necessary for the synthesis of Fe-S clusters and strains lacking this operon have severe defects in a variety of cellular processes. Other cellular loci that impact Fe-S cluster synthesis to a lesser extent have been described. The cyaY locus encodes a frataxin homolog, and it is shown here that lesions in this locus affect Fe-S cluster metabolism. When present in combination with other lesions, mutations in cyaY can result in a strain with more severe defects than those lacking the isc locus.     

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.     

Polyamines are required for virulence in Salmonella enterica serovar Typhimurium

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence.