Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB,yojN or rcsC strains of Salmonella enterica serovar Typhimurium

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.     

Iron regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth

Catecholamines may stimulate enteric bacteria including the foodborne pathogen Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) by two mechanisms in vivo: as a quorum sensing signal and a supplier of iron. To identify genes of Salmonella Typhimurium that respond to norepinephrine, transposon mutagenesis and DNA microarray analysis were performed. Insertional mutations in the following genes decreased norepinephrine-enhanced growth: degS, entE, entF, fes, gpmA, hfq, STM3846. DNA microarray and real-time RT-PCR analyses revealed a decrease in the expression of several genes involved in iron acquisition and utilization during norepinephrine exposure, signifying the iron-limiting conditions of serum-SAPI minimal medium and the siderophore-like activity of norepinephrine. Unlike the wild-type parent strain, growth of neither a fepA iroN cirA mutant nor a fepC mutant, harboring deletional mutations in the outer and inner membrane transporters of enterochelin, respectively, was enhanced by norepinephrine. However, growth of the fepC and the fepA iroN cirA mutants could be rescued by an alternative siderophore, ferrioxamine E, further validating the role of norepinephrine in supplying the organism with iron via the catecholate-specific iron transport system. Contrary to previous reports using small animal models, the fepA iroN cirA mutant of Salmonella Typhimurium colonized the swine gastrointestinal tract, as did the fepC mutant.     

The apeE gene of Salmonella enterica serovar Typhimurium is induced by phosphate limitation and regulated by phoBR

Mutations in apeR, a regulatory locus of the outer membrane esterase apeE from Salmonella enterica serovar Typhimurium, were shown to be alleles of the pstSCAB-phoU high-affinity phosphate transport operon. Expression of apeE was induced by phosphate limitation, and this induction required the phoBR phosphate regulatory system.     

Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium

Aims: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. Methods and Results: Salicylate‐based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate‐based PAE on biofilm‐forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air–liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid‐based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air–liquid interface (top‐forming) and those that form at the surface–liquid interface (bottom‐forming). Conclusion: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. Significance and Impact of the study: Biofilm formation can be prevented through controlled release of nature‐derived antimicrobials formulated into polymer systems.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium

Aims: To analyse genetic changes in the oafA gene explaining the loss of O5‐antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:‐. Methods and Results: The oafA gene in 52 O5‐antigen‐negative and 77 O5‐antigen‐positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:‐ (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5‐antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5‐antigen‐negative strains could be identified. In 77% of the O5‐antigen‐negative strains, a 7‐bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3′end of undetermined size was responsible for the lack of O5‐antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. Conclusions: O5‐antigen‐negative Salm. Typhimurium and Salm. 4,[5],12:i:‐ strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5‐antigen expression. Significance and Impact of the Study: The loss of O5‐antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5‐antigen‐positive and O5‐antigen‐negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i‐) strains.     

The alternative sigma factor sigma is required for resistance of Salmonella enterica serovar Typhimurium to anti-microbial peptides

The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters a variety of anti-microbial peptides during the course of infection. We report here that the extracytoplasmic sigma factor sigma(E) (RpoE) is required for Salmonella resistance to killing by the bactericidal/permeability-increasing protein (BPI)-derived peptide P2 and the murine alpha-defensin cryptdin-4 (Crp4). Moreover, sigma(E)-deficient S. Typhimurium is attenuated for virulence after oral infection of immunocompromised gp91phox(-/-) mice that lack a functional NADPH phagocyte oxidase, suggesting that sigma(E) plays an important role in resistance to non-oxidative mucosal host defences such as anti-microbial peptides. Although both P2 and Crp4 target the cell envelope, bacterial killing by these peptides appears to occur by distinct mechanisms. Formate enhances bacterial resistance to P2, as previously demonstrated, but not to Crp4. Both sigma(E) and cytoplasmic membrane-associated formate dehydrogenase are required for the protective effect of formate against P2. In contrast to P2, Crp4 does not inhibit bacterial respiration at lethal concentrations. However, both peptides induce expression of rpoE, suggesting that they trigger a common mechanism for sensing extracytoplasmic stress.     

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.     

Lesions in gshA (Encoding gamma-L-glutamyl-L-cysteine synthetase) prevent aerobic synthesis of thiamine in Salmonella enterica serovar typhimurium LT2

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.     

The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage

Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.     

The thiamine kinase (YcfN) enzyme plays a minor but significant role in cobinamide salvaging in Salmonella enterica

Cobinamide (Cbi) salvaging is impaired, but not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP:adenosylcobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a DeltacobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycfN(+) allele into a DeltacobU DeltaycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.     

The protective effect of Bdellovibrio‐and‐like organisms (BALO) on tilapia fish fillets against Salmonella enterica ssp. enterica serovar Typhimurium

Aims: To characterize freshwater Bdellovibrio‐and‐like organisms (BALO) isolated in China and examine their potential in controlling growth of Salmonella enterica ssp. enterica serovar Typhimurium on tilapia fillets. Methods and Results: Four BALO isolates were recovered from a pond in Yanzhou of Shandong province, China, with Salm. Typhimurium as prey using double‐layer agar method. Partial 16S rDNA sequencing analysis identified BD2GL, BD5GL and BDXGL as Bdellovibrio bacteriovorus and BD2GS as a Peredibacter sp. Lysis experiments on 32 potentially pathogenic strains revealed that BALO lysis rates are in the range of 56·3–65·6%. On the five Salmonella strains tested, only BD2GS achieved 100% lysis rate. When applied on tilapia fillets against Salm. Typhimurium, BD2GS showed its growth control potential. Cell increments of Salm. Typhimurium were significantly lower (P < 0·05) in two BD2GS‐treated groups compared to control and low‐dose group (BD2GS to prey ratio, 1 : 1) was more effective than high‐dose group (BD2GS to prey ratio, 10 : 1) in controlling Salm. Typhimurium growth. Conclusions: Results of this study indicated that BD2GS could control Salm. Typhimurium growth on tilapia fillets. Significance and Impact of the Study: BALO could be used as a live protective culture in controlling bacterial growth and ensure food safety.     

The crystal structure of dTDP-D-Glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-l-rhamnose pathway

l-Rhamnose is a 6-deoxyhexose that is found in a variety of different glycoconjugates in the cell walls of pathogenic bacteria. The precursor of l-rhamnose is dTDP-l-rhamnose, which is synthesised from glucose- 1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway requiring four enzymes. Significantly this pathway does not exist in humans and all four enzymes therefore represent potential therapeutic targets. dTDP-D-glucose 4,6-dehydratase (RmlB; EC 4.2.1.46) is the second enzyme in the dTDP-L-rhamnose biosynthetic pathway. The structure of Salmonella enterica serovar Typhimurium RmlB had been determined to 2.47 A resolution with its cofactor NAD(+) bound. The structure has been refined to a crystallographic R-factor of 20.4 % and an R-free value of 24.9 % with good stereochemistry.RmlB functions as a homodimer with monomer association occurring principally through hydrophobic interactions via a four-helix bundle. Each monomer exhibits an alpha/beta structure that can be divided into two domains. The larger N-terminal domain binds the nucleotide cofactor NAD(+) and consists of a seven-stranded beta-sheet surrounded by alpha-helices. The smaller C-terminal domain is responsible for binding the sugar substrate dTDP-d-glucose and contains four beta-strands and six alpha-helices. The two domains meet to form a cavity in the enzyme. The highly conserved active site Tyr(167)XXXLys(171) catalytic couple and the GlyXGlyXXGly motif at the N terminus characterise RmlB as a member of the short-chain dehydrogenase/reductase extended family.The quaternary structure of RmlB and its similarity to a number of other closely related short-chain dehydrogenase/reductase enzymes have enabled us to propose a mechanism of catalysis for this important enzyme.     

Reduced transaminase B (IlvE) activity caused by the lack of yjgF is dependent on the status of threonine deaminase (IlvA) in Salmonella enterica serovar Typhimurium

The YjgF/YER057c/UK114 family is a highly conserved class of proteins that is represented in the three domains of life. Thus far, a biochemical function demonstrated for these proteins in vivo or in vitro has yet to be defined. In several organisms, strains lacking a YjgF homolog have a defect in branched-chain amino acid biosynthesis. This study probes the connection between yjgF and isoleucine biosynthesis in Salmonella enterica. In strains lacking yjgF the specific activity of transaminase B, catalyzing the last step in the synthesis of isoleucine, was reduced. In the absence of yjgF, transaminase B activity could be restored by inhibiting threonine deaminase, the first enzymatic step in isoleucine biosynthesis. Strains lacking yjgF showed an increased sensitivity to sulfometruron methyl, a potent inhibitor of acetolactate synthase. Based on work described here and structural reports in the literature, we suggest a working model in which YjgF has a role in protecting the cell from toxic effects of imbalanced ketoacid pools.