Salmonella typhimurium mutants lacking NAD pyrophosphatase.

NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome.     

Sex-determined chemotaxis inSalmonella typhimurium LT2

Chemotactic interaction between F^? and Hfr strains ofSalmonella typhimurium is described. Under the experimental conditions used, the motile Hfr cells are attracted by the F^? cells.     

Mutagenicity of structurally related phenylazo-3-pyridines inSalmonella typhimurium

Studies were conducted to explore structure-activity relationships for 4'-N,Ndimethylamino-1'-phenylazo-3-pyridine and nine structurally related compounds in Salmonella typhimuriunz tester strains TA1535, TA100, TA1537, TA1538, TA98. Each compound was tested for mutagenicity at five or more concentrations that varied from 10-5000 pglplate. We used the standard plate test and the investigations were carried out both in the absence and presence of Aroclor-1254induced rat-liver homogenate and the components of the NADPHgenerating system. Negative response was observed for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and five of its analogues (4'-N,N-diethylamino-1'phenylazo-3-pyridine; 4'-N,N-di-(-hydroxyethylamino)-1'phenylazo-3-pyridine; 4'-N-methylamino sulfonic acid-1'-phenylazo-3-pyrldine; 4'-N,N-dimethylamino-. 6'-acetamido-1'phenylazo-3-pyridine, and 4'-N,N-di-(-hydroxyethylamino)-6'-methyl-1'phenylazo-3-pyridine). When S9 induced by Aroclor-1254 was present, the compound 4'-N,N-dimethylamino-6-methoxy-1'phenylazo-3-pyridine exhibited mulagenic activity in the two strains TA1538 and TA98. The compound 4' ,6'-diamino-3-methyl-1'-phenylazo-3-pyridine was also mutagenic, both in the presence and in the absence of S9 mix. The two compounds 4'-NjVdimethylamino-6-butoxy-1'-phenylazo-3-pyridine and 4'N,N-di-(-hydroxyethylamino)-1'-phenylazo-3-[6-N,N-di-(-hydroxyethylamino)]-pyridine were either weakly mutagenic or nonmutagenic. On the basis of these data, it is concluded that the mutagenicity of phenylazo-3-pyrzdines, like monocyclic aromatic amines and azo dyes, is influenced by the nature of the substifuent chemical groups and their positions in the molecular structure of the compounds.     

Mapping of a gene causing resistance to chlorate inSalmonella typhimurium

Chlorate-resistant mutants ofS. typhimurium LT2 and LT7 and ofS. abony have been isolated, which are deficient in the biosynthesis of nicotinic acid and thiamin and in the fermentation of inositol. These mutants could be divided into 5 groups. The most likely gene order isnicB-chlG-thiB-inlB. This segment is transferred early in conjugation experiments with Hfr H2 and Hfr B2 as donors. In time-of-entry experiments with Hfr B2 as donor the segment entered about 3 minutes afterpur C. Consequently this segment maps in the 79- to 82-minutes region of the genetic map. From recombinant analysis of nic⁺ recombinants obtained in a four-point cross between Hfr B2 and ahis carBpur C del (nic chl G) acceptor the incorporation frequency of the transferred donor fragment was calculated to be about 0.41. The number of crossing-over events per minute length of the chromosome was about the same as in similar crosses betweenE. coli Hfr and F^–. However, between thenic and thepur C markers it was much higher; it may therefore be inferred that there is a higher probability for a crossing-over event in the regions adjacent to the region that is deleted in the recipient.In crosses betweenS. abony Hfr H2 del (nic thi inl chl) and F^– strains no recombinants were observed which have obtained the deletion from the donor. Nearly all auxotrophic or nic⁺ recombinants obtained in a cross between Hfr B2 and a F^– del (nicBthiBchlGinlB) strain have inherited all markers of the donor, which are present in the deletion of the recipient.     

Colicin E1 export inSalmonella typhimurium wild-type and lipopolysaccharide mutants

Colicin export was studied in differentSalmonella typhimurium strains lacking the O-antigen repeating units (O⁻) and different strains with different chemotypes for the lipopolysaccharide core, as well as the wild-type strain (O⁺) to determine the role of lipopolysaccharide length on colicin E1 export. While the lipopolysaccharide length influences the levels of external hemolytic activity inS. typhimurium, no effect was detected on colicin E1 export.     

Absence of alterations in antigenic determinats inSalmonella typhimurium after mecillinam-induced morphological changes

Oval forms ofSalmonella typhimurium resulting from exposure to mecillinam demonstrated electrophoretic differences in the intracellular soluble proteins as compared to the normal organisms. No differences were found, however, in the electrophoretic patterns of cell wall protein.Salmonella rods, or oval forms stained with specific fluorescein-coupled antibodies raised in rabbits against normal or mecillinam-induced abnormal forms, showed identical patterns of fluorescence. Identical lines of precipitins in Ouchterlony system were produced between any combination of antibodies and cytosol protein from normal or mecillinam-exposedSalmonella or with cell wall extracts of either forms. These results indicated that the induced ovoid transformation produced by mecillinam is not accompanied by any demonstrable alterations in the antigenic determinants ofS. typhimurium.     

Phenolic acids reduce the genotoxicity of acridine orange and ofloxacin inSalmonella typhimurium

Naturally occurring plant phenolics,p-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA) and gentisic acid (GA) (25–100 nmol/L) had protective effects on acridine orange (AO; 216 μmol/L)- and ofloxacin (3 μmol/L)-induced genotoxicity inSalmonella typhimurium. FA, GA and CA exhibited a significant concentration-dependent protective effect against the genotoxicity of AO and ofloxacin, with the exception of PA, which at all concentrations tested abolished the AO and ofloxacin genotoxicity. UV spectrophotometric measurements showed the interaction of PA, FA, GA and CA with AO but not with ofloxacin; this interaction is obviously responsible for the reduction of AO-inducedS. typhimurium mutagenicity. In the case of ofloxacin the antimutagenic effect of PA, FA, GA and CA is assumed to be a result of their ability to scavenge reactive oxygen species (ROS) produced by ofloxacin.     

Analysis of chimeric UmuC proteins: identification of regions inSalmonella typhimurium UmuC important for mutagenic activity

UnlikeEscherichia coli, the closely related bacteriumSalmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents. Previous experiments have suggested that these phenotypic differences might result from reduced activity of theS. typhimurium UmuC protein. To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins ofE. coli andS. typhimurium and have constructed a series of plasmid-encoded chimeric proteins. The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon. Constructs expressing mostlyE. coli UmuC were moderately proficient for mutagenesis whereas those expressing mostlyS. typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein ofS. typhimurium is indeed curtailed. The regions responsible for this phenotype were more precisely localized by introducing smaller segments of theS. typhimurium UmuC protein into the UmuC protein ofE. coli. While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212–395 and 396–422 ofE. coli UmuC with those fromS. typhimurium resulted in reduced mutability, while substitution of residues 26–59 caused a dramatic loss of activity. We suggest, therefore, that the primary cause for the poor mutability ofS. typhimurium can be attributed to mutations located within residues 26–59 of theS. typhimurium UmuC protein.     

Structural gene for NAD synthetase in Salmonella typhimurium.

We have identified the structural gene for NAD synthetase, which catalyzes the final metabolic step in NAD biosynthesis. This gene, designated nadE, is located between gdh and nit at 27 min on the Salmonella typhimurium chromosome. Mutants of nadE include those with a temperature-sensitive lethal phenotype; these strains accumulate large internal pools of nicotinic acid adenine dinucleotide, the substrate for NAD synthetase. Native gel electrophoresis experiments suggest that NAD synthetase is a multimeric enzyme of at least two subunits and that subunits from Escherichia coli and S. typhimurium interact to form an active heteromultimer.     

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.     

Regulation of purine biosynthetic genes expression inSalmonella typhimurium IV Oc mutation site ofpurG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein.     

An indispensable gene for NAD biosynthesis in Salmonella typhimurium.

We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme.     

Bifunctional NMN adenylyltransferase/ADP-ribose pyrophosphatase: structure and function in bacterial NAD metabolism

Bacterial NadM-Nudix is a bifunctional enzyme containing a nicotinamide mononucleotide (NMN) adenylyltransferase and an ADP-ribose (ADPR) pyrophosphatase domain. While most members of this enzyme family, such as that from a model cyanobacterium Synechocystis sp., are involved primarily in nicotinamide adenine dinucleotide (NAD) salvage/recycling pathways, its close homolog in a category-A biodefense pathogen, Francisella tularensis, likely plays a central role in a recently discovered novel pathway of NAD de novo synthesis. The crystal structures of NadM-Nudix from both species, including their complexes with various ligands and catalytic metal ions, revealed detailed configurations of the substrate binding and catalytic sites in both domains. The structure of the N-terminal NadM domain may be exploited for designing new antitularemia therapeutics. The ADPR binding site in the C-terminal Nudix domain is substantially different from that of Escherichia coli ADPR pyrophosphatase, and is more similar to human NUDT9. The latter observation provided new insights into the ligand binding mode of ADPR-gated Ca2+ channel TRPM2.     

Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.