Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis

Summary The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). We have isolated, mapped and characterized a large number of Salmonella typhimurium mutants auxotrophic for ALA. These mutants carry defects in either one of two genes, both required for ALA synthesis. The previously identified hemA gene maps at 35 min, and the hemL gene maps at 5 min on the S. typhimurium genetic map. Mutants in hemA and hemL are defective for aerobic and anaerobic respiration, and appear to be oxygen sensitive. The Hem^– phenotype of hemL mutants is less severe than that of hemA mutants. Although hemA and hemL mutants are deficient in heme synthesis, genetic tests indicate that they still synthesize two minor products of the heme pathway, siroheme and cobalamin (vitamin B₁₂), under anaerobic conditions. In contrast, hemB, hemC and cysG mutants, blocked after ALA synthesis, make neither siroheme nor vitamin B₁₂. Double mutants defective in both hemA and hemL also make siroheme. We suggest that hemA and hemL are required for one route of ALA synthesis and that a second, minor route of ALA synthesis may operate in S. typhimurium; this second pathway would be independent of the hemA and hemL functions.Abbreviations Amp ampicillin - Cam chloramphenicol - Kan kanamycin - Tet tetracycline - Str streptomycin - X-gal 5-bromo-4-chloro-3-indolyl--d-galactoside - DES diethyl sulfate     

Cloning of rfaG, B, I, and J genes for glycosyltransferase enzymes for synthesis of the lipopolysaccharide core of Salmonella typhimurium.

R-prime plasmids carrying the pyrE-rfa-cysE region of the chromosome of Salmonella typhimurium were isolated by using the vector pULB113 (RP4::mini-Mu). One of the R-prime plasmids was used as a source of DNA to clone the rfa genes for lipopolysaccharide synthesis to pBR322. The following three hybrid plasmids were constructed: pKZ15, with a 4.0-kilobase EcoRI fragment of S. typhimurium DNA, containing the rfaG gene; pKZ27, a 9-kilobase BglII fragment with the rfaG, rfaB, and rfaI genes; and pKZ26, a 7.7-kilobase HindIII fragment with the rfaG, rfaB, rfaI, and rfaJ genes. We propose that these cloned genes code for four glycosyltransferases used for synthesis of the lipopolysaccharide core region (rfaG for glucosyltransferase I; rfaI for galactosyltransferase I; rfaB for galactosyltransferase II; and rfaJ for glucosyltransferase II). For all four genes, mutants which lacked the appropriate enzyme activity were complemented by the plasmids to give completed core lipopolysaccharide with O (somatic) side chains; for rfaG, rfaB, and rfaI, mutants gave restored or even amplified levels of the appropriate glycosyltransferase in in vitro assays. We show that the order of genes in the region is pyrE-rfaG-(rfaB-rfaI)-rfaJ-rfaL-rfaF -cysE.     

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.     

Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core.

Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes. The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene. It has been designated rfaS. The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein. The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S. typhimurium. Escherichia coli K-12 restriction fragments carrying these genes complement an S. typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant. The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E. coli K-12 have sugar and acceptor specificities different from those of S. typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms. Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome. The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules. Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Antimicrobial action of nisin against Salmonella typhimurium lipopolysaccharide mutants.

The antimicrobial activity of nisin against outer membrane lipopolysaccharide mutants of Salmonella typhimurium LT2 was investigated. Nisin sensitivity was associated with the extent of saccharide deletions from the outer membrane core oligosaccharide. The results indicated that the core oligosaccharide in lipopolysaccharide plays a role in nisin sensitivity.     

Salmonella typhimurium mutants lacking protease II.

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.     

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.     

Mapping of the genetic determinant controlling Salmonella K-antigen synthesis. II. Nonmotile mutants of Salmonella typhimurium

According to the preliminary data, S. typhimurium K-antigen is located in the area of minutes 40-44 on the Salmonella chromosome map. The formation of nonmotile mutants from motile Salmonella strains was induced by the action of nitrosoguanidine. Two main groups of mutants differing in their reaction of agglutination with H- and K-antisera were obtained: Mot-H-K- (motA or motB mutants) and Mot-H-K- (H1- or fla- mutants). The transduction transfer of the sign of motility by phage P22HT to H-K- mutants and to H1- and flaE- mutants led to the restoration of agglutination ability with respect to H- and K-antisera in all Mot+ transductants under study simultaneously. The restoration of H+K+ phenotype was also observed in spontaneous motile revertants obtained from H-K- mutants. Thus, the gene controlling the synthesis of K-antigen in Salmonellae was shown to be incorporated into the Fla operon, the regulatory system of the operon controlling the expression of this gene.     

Temperature-sensitive mutants of RNase E in Salmonella enterica

RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys, Ile207→Ser, Ile207→Asn, and Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.     

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.     

Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants.

The outer membrane layer of the cell wall was isolated from wild-type Salmonella typhimurium LT2 as well as from its mutants producing lipopolysaccharides with shorter saccharide chains. Chemical analysis of these preparations indicated the following. (i) The number of lipopolysaccharide molecules per unit area was constant, regardless of the length of the saccharide side chain in lipopolysaccharide. (ii) In contrast, in "deep rough" (Rd or Re) mutants producing the lipopolysaccharides with very short saccharide chains, the amount of outer membrane protein per unit surface area decreased to about 60% of the value in the wild type. (iii) In the wild type, the amount of phospholipids is slightly less than what is needed to cover one side of the membrane as a monolayer. In comparison with the wild type, the outer membrane of Rd and Re mutants contains about 70% more phospholipids, which therefore must be distributed in both the outer and inner leaflets of the membrane. Freeze-fracture studies showed that the outer membrane of Re mutants were easily fractured, but fracture became increasingly difficult in strains producing lipopolysaccharides with longer side chains. The convex fracture face was always nearly smooth, but the concave fracture face or the outer half of the membrane was densely covered with particles 8 to 10 nm in diameter. The density of particles was decreased in Re mutants to the same extent as the reduction in proteins, suggesting the largely proteinaceous nature of particles. A model for the supramolecular structure of the outer membrane is presented on the basis of these and other results.     

Refined genetic analysis of the region II che mutants in Salmonella typhimurium

Summary From a detailed complementation analysis of the region II che mutants of Salmonella typhimurium, we have located five che genes, cheA, cheW, cheR, cheB, and cheY. We have shown that corrections are required in the previous assignment of the mutations in four strains: both SL2514 and SL2515 which have been reported to be cheY mutants are cheR mutants, SL2539 is not a cheA but a cheW mutant, and ST171 which has been reported to be a cheZ mutant is a double mutant with defects in both cheA and cheB. Since ST171 is the only cheZ mutant so far isolated, the idea that the cheZ gene might play an essential role in chemotaxis in S. typhimurium as in Escherichia coli has lost its experimental basis. Furthermore, a number of deletion mutants in region II resulting from the excision of Tn10 have been isolated and analysed. From these experiments, we propose that the gene order in region II is flaK-flaE-motA-motB-cheA-cheW-cheR-cheB-cheY-flaM-flaC, which is identical with that in E. coli.     

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.