A Refined Map of the hisG Gene of SALMONELLA TYPHIMURIUM

The hisG gene is the most operator-proximal structural gene of the histidine operon; it encodes the feedback-inhibitable first enzyme of the biosynthetic pathway. Previously, hisG mutants were mapped into seven intervals defined by the availble deletion mutations having endpoints in the hisG gene. The map has been refined using over 60 new deletion mutants. The new map divides the gene into 40 deletion intervals, which average approximately 30 base pairs in length. The map has been used to analyze the distribution of insertion sites for the transposable element Tn10 and has permitted conclusions on the diistribution of duplication endpoints. The map promises to be useful in analysis of his regulation and, more particularly, in the determination of the possible role of the hisG enzyme in this mechanism.     

Ochre Suppression in SALMONELLA TYPHIMURIUM

A bacterial strain was constructed which permitted positive selection for ochre suppressor mutations as well as for the loss of suppressor function. A derivative bearing an ochre suppressor mutation was selected following mutagenesis with N-methyl-N-nitroso-N'-nitroguanidine. The suppressor-bearing strain was treated with nitrous acid to eliminate suppressor function by mutation, and a strain lacking suppressor activity was selected. The selected strain which had lost suppressor function was then subjected to mutagenesis to induce a second suppressor mutation. The alternating sequence (induction of an ochre suppressor mutation → induction of a mutation eliminating ochre suppressor activity) was repeated 29 and one-half times in a single strain. Some of the suppressor mutations were tentatively mapped at four locations on the chromosome. The first suppressor mutation selected maps at about minute 30 on the chromosome. The second suppressor selected maps at approximately minute 60, while the third suppressor maps nearby, possibly as far as minute 72. Among the subsequently selected suppressor mutations, all eleven which were mapped were cotransducible with the gal and nic loci near minute 36 on the chromosome and may represent more than one suppressor gene. Deletions were selected which inactivate two of the ochre suppressor alleles mapping near the gal-nic region, suggesting that one or more such genes are dispensable. Some evidence also suggests that the occurrence of either deletion mutations or transduction-mediated recombination events in the gal-nic region can cause instability of nearby suppressor alleles.     

Nonsense Motility Mutants in SALMONELLA TYPHIMURIUM

Of 313 motility-deficient mutants isolated from an LT2 his(amber) strain fixed in phase 1 by gene vh2(-), 25 regained motility when amber or ochre suppressors were introduced, in F' factors or by transduction. The fla mutants (23 amber, 1 ochre) fell in complementation groups A, B, C, F, K, a new group, M, and at least one further new group; the hypothesis of a fla gene which specifies only an RNA structural component of a flagellum-synthesizing basal apparatus is disproven for the corresponding genes. Hfr and transductional crosses confirmed gene assignments from complementation and indicated that flaM and another new fla locus map near H1. A small minority of motile bacteria were detectable in many of the amber fla mutants. In groups A and F some pairs of amber fla mutants complemented each other, and perhaps each of these groups corresponds to more than one structural gene. The suppressed derivatives of a mutant with an amber mutation in H1 made flagella morphologically and serologically indistinguishable from wild-type flagella. A slow-spreading but flagellate mutant showed mainly non-translational motility in broth, and in a viscous medium the bacteria reversed very frequently; its amber mutation, probably near H1, is inferred to cause a defect in chemotaxis, so that the bacteria give the avoidance reaction continuously.     

Selection for a Large Genetic Duplication in SALMONELLA TYPHIMURIUM

Salmonella typhimurium strains containing a duplication of nearly a third of the genome have been isolated by a simple procedure involving selection for improved utilization of L-malate as sole carbon source. The duplication occurs at a very high spontaneous frequency. Strains containing the duplication can be isolated selectively on malate medium, or by a non-selective procedure involving Hfr conjugation. When strains containing the duplication are maintained on non-selective medium, the duplication is readily lost. Genetic evidence suggests that the duplication is chromosomal and tandem. The fact that the recA gene is included in the duplication has been used to obtain evidence that the recA1 marker is recessive to its wild-type allele. Unlike tandem duplications previously described in E. coli, the duplication described in this report appears to have unique endpoints.     

New Suppressors of Frameshift Mutations in SALMONELLA TYPHIMURIUM

Several new types of suppressor mutants have been isolated. These were identified among revertants of mutants originally generated by mutagens other than the acridine-derived ICR191. The new suppressors correct mutations other than those with runs of C or G which are recognized by the previously described suppressors. Several frameshift mutations are corrected by more than one suppressor type. Apparently, the DNA base sequence near these mutant sites includes sites of action for several distinct suppressor types.     

Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

Selection of SALMONELLA TYPHIMURIUM Mutants with Altered Serine Transhydroxymethylase Regulation

In Salmonella typhimurium the glyA gene product, serine transhydroxymethylase (E.C. 2.1.2.1.; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible for the interconversion of serine and glycine. This reaction also provides the cell with one-carbon units from the 5,10-methylene-tetrahydrofolate formed during glycine synthesis. Despite the importance of this enzyme, however, no mutants in which its regulation has been specificially altered have been isolated. To isolate such mutants, we have devised a selection procedure using a strain (glyA951) in which the serine transhydroxymethylase activity is reduced. When this enzyme is completely repressed, the mutant requires gylcine for growth. Revertants which retain the glyA951 lesion, but no longer require glycine, have been isolated and the serine transhydroxymethylase regulation examined. One revertant has a 7-fold elevated serine transhydroxymethylase level, which can be repressed the normal amount (about 5-fold) when the cells are grown in supplemented media. Another revertant has only a 2-fold higher serine transhydroxymethylase level; however, the amount of repression is reduced. The new lesions in both mutants cotransduce with the glyA gene and are distinct from other mutations that alter the regulation of both serine transhydroxymethylase and the methionine biosyntheitc enzymes.     

Genetic Characterization of the SufJ Frameshift Suppressor in SALMONELLA TYPHIMURIUM

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.--The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.--Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA).     

Characterization of SALMONELLA TYPHIMURIUM Mutants with Altered Glutamine Synthetase Activity

A number of glutamine auxotrophs of Salmonella typhimurium were isolated and characterized genetically. Three of the mutations appear to be closely linked and are complemented by episomes carrying the glnA region of Escherichia coli. The lesions in these strains are approximately 20% linked by P1 transduction with a mutation in the rha gene, but are unlinked to ilv. Another mutation causing glutamine auxotrophy in strain JB674 is genetically distinct from the others. Strain JB674 grown in glucose medium containing ammonia as the nitrogen source has reduced levels of glutamine synthetase that is more adenylylated than in the parent strain, suggesting that the enzyme can not be deadenylylated normally. The lesion causing glutamine auxotrophy in JB674 lies in the region corresponding to the glnB and glnE genes affecting glutamine synthetase modification in Klebsiella areogenes. Four Gln+ revertants of JB674 have glutamine synthetase activities 4 to 6 fold higher than normal. One mutation causing this increased enzyme synthesis has been shown by three-factor crosses with the glnA mutations to lie near or within the glnA gene.     

Conditionally Expressed Missense Mutations: The Basis for the Unusual Phenotype of an Apparent trpD Nonsense Mutant of SALMONELLA TYPHIMURIUM

Tryptophan auxotroph trp-28 is anomalous since preliminary mapping and suppression studies indicate the presence of a single amber nonsense mutation either late in trpE or early in trpD, but enzymological tests indicate the complete inactivation of both genes in this strain. Since the trpE and trpD genes are contiguous and encode the two subunits of a multifunctional enzyme complex, it was of interest to learn the mechanism of action of this apparent pleiotropic nonsense mutation. Our study has revealed that the phenotype of this strain derives not from a single mutation, but from the presence and interaction of multiple mutations. Besides the recognized amber mutation (designated trpD28), this strain carries two additional, conditionally expressed missense mutations (designated trpE1651 and trpD1652). The trpD28 amber codon maps in the promoter-proximal region 1 of trpD and eliminates the glutamine amidotransferase activity of the bifunctional trpD polypeptide. The trpD1652 mutation maps in the promoter-distal region 2 of trpD and severely reduces (but does not eliminate) the phosphoribosyl transferase activity of the trpD polypeptide. The trpE1651 mutation maps in the anterior part of trpE and causes a rapid loss of activity of the trpE polypeptide, but only when it exists as an uncomplexed subunit. The existence of the two missense mutations escaped prior notice in standard recombinational tests since the nature of each mutation is such that neither is detectable by the nutritional screens normally used in such tests unless an unsuppressed chainterminating mutation, such as trpD28, is also present.