Salmonella typhimurium mutants generally defective in chemotaxis.

The mutations of eight chemotaxis-deficient strains of Salmonella typhimurium, including five new mutants in strain LT2, were mapped by P22 transduction in relation to various fla mot deletions in S. abortus-equi. Seven recessive che mutations mapped between motB and flaC: three, all nontumbling, the che region I, adjacent to motB, and four, including one ever-tumbling, in che region II, adjacent to flaC. Mutant che-107, never-tumbling and dominant to wild type, mapped at flaAII, other mutations of which cause either absence of flagella or lack of locomotor function. We surmise that gene flaAII specifies a protein that polymerizes to form an essential component of the basal apparatus (so that absence of gene product prevents formation of flagela); that a component built up from certain mutationally altered proteins cannot transmit (or generate) active rotation of the hook and flagellum, and so causes the Mot (paralysis) phenyotype; and that a component built up from protein with the che-107 alteration permits only counterclockwise rotation, so that the tumble, normally produced by transient clockwise rotation, cannot be effected.     

Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-¹⁴C]riboflavin. The vitamin B₁₂ isolated was ¹⁴C-labeled. It was shown by chemical degradation that the ¹⁴C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium. Received: 25 August 1998 / Accepted: 27 October 1998     

Salmonella typhimurium mutants defective in the formate dehydrogenase linked to nitrate reductase.

Six fdn mutants of Salmonella typhimurium defective in the formation of nitrate reductase-linked formate dehydrogenase (FDHN) but capable of producing both the hydrogenase-linked formate dehydrogenase (FDHH) and nitrate reductase were characterized. Results of phage P22 transduction experiments indicated that there may be three fdn genes located on the metE-metB chromosomal segment and distinct from all previously identified fdh and chl loci. All six FDHH+ FDHN- mutants were found to make FDHN enzyme protein which was indistinguishable from that of the wild type in electrophoretic studies. However, the results of the spectral studies indicated that all six mutants were defective in the anaerobic cytochrome b559 associated with FDHN. All contained the cytochrome b559 associated with nitrate reductase in amounts equal to or greater than the wild type. The results of the transduction experiments also indicated that the metE- metB segment of the Salmonella chromosome resembles that of Escherichia coli more than was originally thought.     

Salmonella typhimurium mutants defective in acetohydroxy acid synthases I and II.

An analysis of transposon-induced mutants shows that Salmonella typhimurium possesses two major isozymes of acetohydroxy acid synthase, the enzymes which mediate the first common step in isoleucine and valine biosynthesis. A third (minor) acetohydroxy acid synthase is present, but its significance in isoleucine and valine synthesis may be negligible. Mutants defective in acetohydroxy acid synthase II (ilvG::Tn10) require isoleucine, alpha-ketobutyrate, or threonine for growth, a mutant defective in acetohydroxy acid synthase I (ilvB::Tn5) is a prototroph, and a double mutant (ilvG::Tn10 ilvB::Tn5) requires isoleucine plus valine for growth.     

Isolation and characterization of lon mutants in Salmonella typhimurium.

In this paper we report the isolation and characterization of lon mutants in Salmonella typhimurium. The mutants were isolated by using positive selection by chlorpromazine resistance. The physiological and biochemical properties of the lon mutants in S. typhimurium are very similar to those of Escherichia coli lon mutants. Mutants altered at this locus contain little or no activity of the ATP-dependent protease La and show a number of pleiotropic phenotypes, including increased production of capsular polysaccharides, increased sensitivity to UV light and other DNA-damaging agents, and a decreased ability to degrade abnormal proteins.     

Salmonella typhimurium mutants defective in UDP-D-galactose:lipopolysaccharide alpha 1,6-D-galactosyltransferase. Structural, immunochemical, and enzymologic studies of rfaB mutants

The biochemical defect in a class of Salmonella typhimurium mutants (rfaB) defective in biosynthesis of the lipopolysaccharide core is described. Structural, immunochemical and enzymologic studies showed that: (i) the core polysaccharide completely lacked the branch alpha 1,6-D-galactosyl residue of the normal lipopolysaccharide as shown by methylation analysis and 1H nmr spectroscopy; (ii) the mutant lipopolysaccharides acted as acceptors for transfer of D-galactose from UDP-D-galactose into alpha 1,6 linkage to the proximal D-glucosyl residue of the core in a reaction catalyzed by an enzyme activity present in extracts from rfaB+ cells; (iii) the UDP-D-galactose:(glucosyl)lipopolysaccharide alpha 1,6-D-galactosyltransferase activity was absent from extracts of rfaB cells.     

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5''-phosphate synthesis.

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.     

Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide: 5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin. Surprisingly, CobT can phosphoribosylate a wide range of aromatic substrates, giving rise to a wide variety of lower ligands in cobamides. To understand the molecular basis for this lack of substrate specificity, the x-ray structures of CobT complexed with adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, p-cresol, and phenol were determined. Furthermore, adenine, 5-methylbenzimidazole, 5-methoxybenzimidazole, and 2-hydroxypurine were observed to react with NaMN within the crystal lattice and undergo the phosphoribosyl transfer reaction to form product. Significantly, the stereochemistries of all products are identical to those found in vivo. Interestingly, p-cresol and phenol, which are the lower ligand in Sporomusa ovata, bound to CobT but did not react with NaMN. This study provides a structural explanation for how CobT can phosphoribosylate most of the commonly observed lower ligands found in cobamides with the exception of the phenolic lower ligands observed in S. ovata. This is accomplished with minor conformational changes in the side chains that constitute the 5,6-dimethylbenzimidazole binding site. These investigations are consistent with the implication that the nature of the lower ligand is controlled by metabolic factors rather by the specificity of the phosphoribosyltransferase.     

Isolation and characterization ofAcinetobacter sp. mutants defective in exopolysaccharide biosynthesis

Nitrosoguanidine-induced mutants ofAcinetobacter sp. defective in exopolysaccharide biosynthesis did not differ from the parent strain in distinguishing physiological and biochemical properties, such as requirements for growth factors, utilization of mono- and disaccharides, and resistance to antibiotics. The genetic relation of parent and mutant strains was shown by 16S rRNA PCR analysis. The comparative study of parent and mutant strains with respect to resistance to unfavorable environmental factors confirmed our hypothesis thatAcinetobacter sp. exopolysaccharides perform protective functions. Hybridization experiments revealed the conjugal transfer of plasmid R68.45 fromPseudomonas putida BS228 (R68.45) to mutant but not to the parentAcinetobacter sp. strains. The role of theAcinetobacter sp. exopolysaccharides in providing the genetic stability of this bacterium is discussed.     

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.     

Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin

A number of mutants of Salmonella typhimurium were isolated which are blocked in the biosynthesis of enterobactin, an iron chelator that is secreted by the wild-type bacteria when they are grown on low iron media. One class of these enb mutants accumulates the enterobactin precursor 2,3-dihydroxybenzoic acid, and another class does not accumulate any detectable catechol precursor. The enb mutants grow very well on a glucose-mineral salts medium. Addition of citrate, itself an iron chelator, to the medium drastically inhibits growth unless the medium is supplemented with enterobactin or iron salts. Citrate inhibits iron uptake from the medium by enb mutants; enterobactin counteracts this inhibition and also, by itself, increases iron uptake. Thus, the apparent function of enterobactin is to promote the absorption of iron from the medium by the bacteria. Transduction experiments showed that the genes for enterobactin biosynthesis are closely linked on the S. typhimurium chromosome. It is suggested that they form an operon which is repressed by the presence of iron. S. typhimurium can utilize the iron chelate ferrichrome. (Deferriferrichrome is a cyclic hexapeptide that is produced by some fungi but not by S. typhimurium.) The enb mutants use ferrichrome as an effective growth factor.     

Release of flagellar filament-hook-rod complex by a Salmonella typhimurium mutant defective in the M ring of the basal body.

A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.     

The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.