Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.

Previous studies of the phase 1 flagellar filament protein (flagellin) in strains of five serovars of Salmonella indicated that the central region of the fliC gene encoding the antigenic part of the protein is hypervariable both between and within serovars. To explore the possible use of this variation as a source of information on the phylogenetic relationships of closely related strains, we used the polymerase chain reaction technique to sequence part of the central region of the phase 1 flagellar genes of seven strains of Salmonella typhimurium that were known to differ in chromosomal genotype, as indexed by multilocus enzyme electrophoresis. We found that the nucleotide sequences of the central region were identical in all seven strains and determined that both the previously published sequence of the fliC gene in S. typhimurium LT2 and a report of a marked difference in the amino acid sequence of the phase 1 flagellins of two isolates of this serovar are erroneous. Our finding that the fliC gene is not evolving by sequence drift at an unusually rapid rate is compatible with a model that invokes lateral transfer and recombination of the flagellin genes as a major evolutionary process generating new serovars (antigen combinations) of salmonellae.     

Sequence of the phosphomannose isomerase-encoding gene of Salmonella typhimurium

The pmi gene, encoding phosphomannose isomerase, of Salmonella typhimurium, was cloned in Escherichia coli K-12, and the protein product visualised in minicells. The cloned gene was sequenced; there was 77.4% nucleotide homology between the cloned pmi gene and the analogous manA gene of E. coli K-12, and 86.2% amino acid sequence homology between their presumptive gene products.     

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.     

Peptidase mutants of Salmonella typhimurium

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.     

rfaP mutants of Salmonella typhimurium

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.     

Genetic analysis of carbohydrate transport-deficient mutants of Salmonella typhimurium

Mutants (car) isolated from Salmonella typhimurium were unable to utilize or ferment the following carbohydrates (all d-configuration): glucose, fructose, mannose, N-acetylglucosamine, sorbitol, mannitol, maltose, melibiose, and glycerol. The mutants did utilize galactose, glucose 6-phosphate, gluconic acid, glucuronic acid, pyruvate, and l-lactate. Biochemical analysis showed that there were two classes of mutants, each lacking one component of a phosphotransferase system. CarA mutants were deficient in enzyme I; carB lacked the phosphate carrier protein, HPr. Mapping experiments showed that the carA gene was located near pro; the carB gene mapped near purC.     

Translation inhibition of the Salmonella fliC gene by the fliC 5' untranslated region, fliC coding sequences, and FlgM

The 5'-untranslated region (5'UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5' end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (-38C:U) in the fliC 5'UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the -38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5'UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the -38C:T motility defect, while flgM mutants defective in anti-sigma28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5'UTR (-15G:A) results in a translation defect by itself but, in combination with the -38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.     

Deoxynucleoside-sensitive mutants of Salmonella typhimurium

Summary Thymineless mutants ofSalmonella typhimurium which are able to grow with low added concentrations of thymine (20 M) fall into two classes on the basis of growth on deoxyribose as sole carbon source. Those which can grow are deoxyribomutase negative and those which cannot are deoxyriboaldolase negative. The former class are inhibited by deoxynucleosides and this provides a method for discriminating between different classes oftlr mutants ofEscherichia coli K12, which cannot utilize deoxyribose as a carbon source. It is suggested that the sensitivity of deoxyriboaldolase negative strains is due to the accumulation of deoxyribose-5-phosphate. The data also indicate that deoxyribose-5-phosphate is the inducer of thymidine phosphorylase. It seems that one or both of the deoxyribose phosphates is the toxic compound, and that reversal of inhibition by ribonucleosides is due to inhibition of the enzymes catalysing their formation from deoxynucleosides. We propose that the symbolsdrm anddra be used to denote the structural genes for deoxyribomutase and deoxyriboaldolase respectively.     

Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella

Non-flagellate H2 mutants were isolated from a phase-2 stable strain, SJW806 H1-gt- H2-enxon vh2-, a derivative of Salmonella typhimurium. By transductional crosses a deletion map and a recombination map of the H2 gene were made. There are three regions especially rich in nonflagellate mutational sites. By the use of the deletion map, mutational sites of 21 flagellar shape mutants were also determined. Most of them were located at two regions which coincide with two of the three regions rich in non-flagellate mutational sites. A gene, vh2, is closely linked to the promoter side of the H2 gene. Three-factor transductional crosses showed that the vh2 gene was on the left of the H2 gene in the present map. The H2 gene forms part of an operon with the distal gene rh1 which specifies the H1 repressor. Thus, a polarity effect of the H2 mutations on the expression of the rh1 gene was examined by observing whether a wild-type H1 allele introduced into the H2 mutants was expressed or not. Many of the H2 mutations were polar, and most of the strongly polar mutations were located in the left (promoter-proximal) half of the H2 gene, while most of the mutations in the right half of the gene were weakly polar or non-polar.     

Isolation and genetic analysis of operator-constitutive mutants of the H1 operon in Salmonella typhimurium

In phase-2 cells of diphasic Salmonella strains, expression of H1 is repressor, coded for by the rh1 gene. A procedure for the isolation of operator-constitutive (H1-Oc) mutants of the H1 operon is described. Using three-factor crosses between an H1-Oc H1 strain and H1-O+ H1 strains, where motility recovery via H1-phase (or phase 1) flagellation was used as the selected marker and the H1-O character was the unselected marker, the relative position of the H1-Oc site to the H1 gene was determined. A diphasic H1-Oc strain produced, in phase 2, copolymer filaments composed of H1 and H2 flagellin.     

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.     

Oligopeptidase-deficient mutants of Salmonella typhimurium.

An oligopeptidase that hydrolyzes N-acetyl-L-alanyl-L-alanyl-L-alanyl-L-alanine (AcAla4) has been identified in extracts of Salmonella typhimurium. Mutants lacking this activity have been isolated in dcp mutant strains by screening extracts of mutagenized clones for failure to hydrolyze AcAla4 or by screening colonies for inability to use AcAla4 as a nitrogen source. Double mutants (dcp optA) lacking both oligopeptidase A and dipeptidyl carboxypeptidase cannot use AcAla4 as a nitrogen source, although dcp+ optA and dcp optA+ strains grow on this peptide. The mutations responsible for the loss of activity map at a locus (optA) between asd (75 map units) and xylA (78 map units). Oligopeptidase A hydrolyzes certain N-blocked tetrapeptides, unblocked pentapeptides, and unblocked hexapeptides, usually but not always liberating the C-terminal tripeptide. These two activities seem to be responsible for the production of a large fraction of the dipeptides that accumulate during protein breakdown in a pepN pepA pepB pepD strain.