Characterization of a cold-sensitive hisW mutant of Salmonella typhimurium

Previous studies of hisW mutants of Salmonella typhimurium have led to the suggestion that such strains are defective in tRNA maturation. (J. E. Brenchley and J. Ingraham, J. Bacteriol. 114:528-536, 1973). In this study, we report that one hisW strain is defective in the accumulation of all stable RNA species. Polyacrylamide gel electrophoresis of radiolabeled RNA indicated tha at the nonpermissive temperature (20 degrees C) all stable RNa species in the cold-sensitive hisW3333 mutant were synthesized and rapidly degraded. We propose that the cold sensitivity of this strain is caused by such a restriction in stable RNA accumulation at low temperature. In vitro and in vivo studies demonstrated that the RNA degraded in this strain was synthesized de novo and was not preexisting RNA. Furthermore, physiological and genetic recovery from the cold-sensitive hisW phenotype resulted in relatively normal RNA synthesis and accumulation. Thus, the RNA alterations observed in this strain were not explained by defects in a tRNA modification enzyme. Rather, these findings suggest the existence of defective RNA processing and that a control mechanism for the overall synthesis or accumulation of stable RNA species is altered in the hisW3333 mutant.     

Cold-Sensitive Mutant of Salmonella typhimurium Defective in Nucleosidediphosphokinase

A cold-sensitive mutant of Salmonella typhimurium defective in nucleosidediphosphokinase (ATP:nucleosidediphosphate phosphotransferase, EC 2.7.4.6) has been isolated and characterized. The mutant contains only 2% of the enzyme activity found in the parent, and the heat lability of this activity is 10 times that from the parent at 33 C. Mutant extracts lack the ability to convert any of 11 nucleoside diphosphates tested to the corresponding nucleoside triphosphates, but the nucleosidemonophosphatase activities are normal. Although the nucleoside triphosphate pools of the mutant are depressed significantly at the restrictive temperature (20 C), they are slightly elevated at the permissive temperature (37 C). The levels of guanosine pentaphosphate and guanosine tetraphosphate are dramatically increased. Two representative enzymes of pyrimidine de novo synthesis, aspartic transcarbamylase and dihydroorotate dehydrogenase, are fully repressed at both 37 and 20 C. Intracellular pools of uridine diphosphate are depressed at both permissive and restrictive temperature.     

Osmotic-Sensitive Mutant of Salmonella typhimurium

A strain (DA82) having peculiar osmotic properties was isolated in Salmonella typhimurium. The mutant shows increased elasticity of its cell wall and makes spherical instead of elongated cells, regardless of the osmolality of the medium. The strain withstands dilution in distilled water without disruption or death and grows normally in 0.1 molal NaCl broth (240 milliosmol), but it dies exponentially in low-osmolality broth (40 milliosmol). Addition of salts or sucrose instantly stops death and allows growth and cell division to proceed. Death is not due to lysis because this appears at later times and at a much lower rate. Osmotic inactivation is temperature-dependent: higher death rates occur at higher incubation temperatures. Inhibition of protein synthesis by chloramphenicol (20 mug/ml) prevents osmotic death. At 37 C and at lower temperatures, the phenomenon of osmotic death is transient. After a variable interval, growth of the osmotic-sensitive strain resumes. It is assumed that the strain's osmotic behavior is due to membrane defectiveness. The membrane disfunction and the wall defect shown by the strain may be consequences of a single genetic alteration or the results of independent mutations.     

Round-Cell Mutant of Salmonella typhimurium

A mutation at the divD locus in Salmonella typhimurium confers a round-cell morphology and enhances cell division under certain growth conditions.     

Altered regulation of isoleucine-valine biosynthesis in a hisW mutant of Salmonella typhimurium.

Control of isoleucine-valine biosynthesis was examined in the cold-sensitive hisW3333 mutant strain of Salmonella typhimurium. During growth at the permissive temperature (37 degrees C), the isoleucine-valine (ilv) biosynthetic enzyme levels of the hisW mutant were two- to fourfold below these levels in an isogenic hisW+ strain. Upon a reduction in growth temperature to partially permissive (30 degrees C), the synthesis of these enzymes in the hisW mutant was further reduced. However, synthesis of the ilv enzymes was responsive to the repression signal(s) caused by the addition of excess amounts of isoleucine, valine, and leucine to the hisW mutants. Such a "super-repressed" phenotype as that observed in this hisW mutant is similar to that previously shown for the hisU1820 mutant, but was different from the regulatory response of the hisT1504 mutant strain. Moreover, by the use of growth-rate-limiting amounts of the branched-chain amino acids, it was shown that this hisW mutant generally did not increase the synthesis of the ilv enzymes as did the hisW+ strain. Overall, these results are in agreement with the hypothesis that the hisW mutant is less responsive to ilv specific attenuation control than is the hisW+ strain and suggest that this limited regulatory response is due to an alteration in the amount or structure of an element essential to attenuation control of the ilv operons.     

Neurospora crassa Cytoplasmic Ribosomes: Isolation and Characterization of a Cold-Sensitive Mutant Defective in Ribosome Biosynthesis

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.     

Growth Inhibition by Hexoses of a Temperature-Sensitive Thiazoleless Mutant of Salmonella typhimurium

A Salmonella typhimurium mutant showing impairment in the utilization of hexoses was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. At 30 C, it grew with hexoses (glucose, fructose, galactose, mannitol), glycerol, succinate, or acid-hydrolyzed casein. At 37 C, it failed to grow with any of the hexoses. Enzymatic determinations demonstrated, however, that the enzymes of the glycolytic pathway (up to the formation of triose phosphates) were present and active at 25 and 32 C. At 42 C, the mutant did not grow with any of the carbon sources used. At both 37 and 42 C, the mutant grew perfectly well with hexoses if yeast extract was present. The metabolite required for growth was thiamine or, specifically, its thiazole moiety. If glucose was added to a culture growing in glycerol, at 37 C, growth was inhibited. This inhibition was relieved by the addition of thiamine or thiazole. Thus, at 37 C and only in the presence of hexoses, the mutant manifests a requirement for thiazole. This auxotrophy is absolute at 42 C. These data indicate that, in this mutant, some derivative of hexoses inhibits the synthesis of thiazole, and that this inhibition is also dependent on the temperature of incubation. The position in the bacterial chromosome of the genetic locus of this lesion (thz(-)) was determined by conjugation and found to coincide with the only thiamine (thi) locus so far reported.     

Mutant strains of Salmonella typhimurium with defective phosphoribosylpyrophosphate synthetase activity

A mutant of Salmonella typhimurium with undetectable phosphoribosylpyrophosphate (PRPP) synthetase activity in vitro and abnormally low PRPP pools in vivo was identified by screening temperature-sensitive isolates by an autoradiographic procedure. The lack of PRPP synthetase activity in vitro and temperature-sensitive growth were shown to result from separate, but closely linked mutations mapping at 47 units on the Salmonella chromosome. Mutant cell extracts prepared by a variety of methods did not show any detectable PRPP synthetase activity, but material that was immunochemically cross-reactive with PRPP synthetase was detected by complement fixation analysis. A second mutant, isolated by localized mutagenesis, contained about half the PRPP synthetase and cross-reacting material of the parental strain.     

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.     

Properties of a Salmonella typhimurium Mutant with an Incomplete Deficiency of Uridinediphosphogalactose-4-Epimerase

A galactose-negative mutant, nonleaky in respect to fermentation and utilization, isolated from a smooth Salmonella typhimurium strain by phage selection and inferred deficient of uridine diphosphate (UDP)-galactose-epimerase, was used for experiments on relation of somatic lipopolysaccharide (LPS) character to virulence. Extracts of induced mutant cells retained ca. 1% of wild-type epimerase activity and had only ca. 5% of wild-type kinase and uridyl transferase activities; also, some cultural properties of the mutant differed from those of mutants with complete defects of epimerase only. The mutant was not galactose sensitive, presumably because of its kinase defect. Although the mutant had the phage pattern (including C21-sensitivity) of an epimerase mutant, it was susceptible to transduction by phage P22 and was O-agglutinable, even when grown on defined medium; its LPS must therefore contain some O polymer, including endogenous galactose, resulting from residual epimerase activity. Growth on galactose-supplemented medium restored smooth phage sensitivity; since the mutant was partly inducible this may result, at least in part, from increased endogenous production of UDP-galactose. The mutant was made galactose positive by introduction of an F'-gal(+) plasmid. Base-change and frame-shift mutagens did not increase the frequency of reversion above the spontaneous rate. An insertion into the operator-promoter region of the gal operon seems the most likely mechanism of the mutation.     

Integration and Induction of Phage P22 in a Recombination-deficient Mutant of Salmonella typhimurium

Phage P22 can integrate as prophage into a recombination-deficient (Rec(-)) strain of Salmonella typhimurium. At 37 C, the integration efficiency is only 10% that in Rec(+) infection, but at 25 C the efficiencies in Rec(-) and Rec(+) hosts are similar. Rec(-) lysogens cannot be induced by ultraviolet irradiation or by treatments with the chemical inducing agents streptonigrin or mitomycin C. Heat induction of Rec(-) cells lysogenic for a temperature-sensitive c(2) mutant (ts c(2)) is normal, showing that the Rec(-) cell has the machinery necessary for prophage excision. Ultraviolet irradiation of Rec(-) (ts c(2)) lysogens prior to heat induction does not prevent the formation of infective centers after temperature shift. Thus, the noninducibility of Rec(-) lysogens is not due to destruction of the prophage as a result of ultraviolet irradiation. Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization experiments demonstrate that no increase in phage-specific RNA synthesis occurs after ultraviolet irradiation of a Rec(-) (c(+)) lysogen. The Rec(-) mutant appears to lack part of the mechanism required to destroy the phage repressor and allow the initiation of early phage functions such as messenger RNA synthesis. A similar conclusion was reached previously for an Escherichia coli Rec(-) strain.     

Mutant of Salmonella typhimurium LT2 deficient in DNA adenine methylation.

A mutant of Salmonella typhimurium LT2 deficient in methylation of the adenine residues in the sequence 5'-GATC-3' was isolated. The mutation (dam-1) was linked to the cysG locus, and the properties of the mutant were similar to those of Escherichia coli dam mutants. Reversion of the hisC3076 frameshift marker by 9-aminoacridine was substantially enhanced by the dam-1 mutation, implying a direct role for adenine methylation in the prevention of frameshift mutation induction.     

鼠伤寒沙门菌spvB基因缺陷突变株的构建

高度保守的spvB基因,其编码产物具有ADP核糖基转移酶活性,是导致受染后细胞发生凋亡的重要毒力因子.为进一步研究该基因致病机制,构建了鼠伤寒沙门菌spvB基因缺陷突变株.根据GenBank鼠伤寒沙门菌spvB基因序列,用Primer Premier 5.0设计PCR特异性引物,获得spvB基因缺陷性核苷酸片段后连接至自杀质粒PGMB151.将构建的自杀载体导入含有spvB基因的鼠伤寒沙门菌标准株SR-11中进行同源重组,PCR筛选缺陷突变株.经PCR和DNA序列测定,成功获得了spvB基因缺陷的鼠伤寒沙门菌突变株.     

Mutant of Salmonella typhimurium That Channels Infecting Bacteriophage P22 Toward Lysogenization

A slowly growing, polymyxin-sensitive mutant of Salmonella typhimurium was isolated. Wild-type phage P22 form plaques on the mutant at 5 x 10(-4), the frequency observed on wild-type hosts. All P22 clear mutants form plaques with near normal frequency. The inability of the mutant to form plaques is correlated with an increase in lysogenization frequency. The cause of the increased lysogenization frequency is not known, but it is not the result of overproduction of cyclic adenosine 5'-monophosphate.     

Characterization and cloning of enterotoxin genes of Salmonella typhimurium

Five of fifty five strains of Salmonella typhimurium of human origin was hybridized with both the LT-A and LT-B gene of Escherichia coli. The remarkably erythromatous and indurated response on rabbit skin and significant elongation of Chinese Hamster Ovary (CHO) cells indicated the production of enterotoxin of these isolates. The Salmonella enterotoxin is heat-labile and is not a secretory product. The LT gene of E. coli was used to analyze the chromosome and plasmid DNA from Salmonella typhimurium strains for toxin gene sequences. Southern blot analysis demonstrated that the toxin gene was located on the plasmid but not on the chromosome. Restriction enzymes BamHI, EcoRI, HindIII and PstI were used to analyze the DNA isolated from salmonella strains Nos.22, 52, 55 and 59. Three DNA fragments with size of 5.2 Kb of strain 22, 5.0 Kb of strain 52 and 8.6 Kb of strain 59 were identified as containing the enterotoxin gene. Plasmid pUC19 was used as the vector to clone these DNA fragments in E. coli. The rabbit skin permeability test indicated that Salmonella enterotoxin could be synthesized at readily detectable levels in these transformed E. coli.     

Characterization of Constitutive Galactose Permease Mutants in Salmonella typhimurium

Salmonella typhimurium strains, lacking both enzyme I and the phosphocarrier protein, HPr, of the phosphoenolpyruvate-sugar phosphotransferase system, cannot transport or metabolize glucose and other sugar substrates of this enzyme system. Mutants which regain the ability to specifically utilize glucose were found to constitutively synthesize a galactose permease by virtue of a mutation in the galR gene. This permease, shown to be an active transport system, does not require HPr or enzyme I for activity.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.     

Characterization of amber and ochre suppressors in Salmonella typhimurium.

Amber and ochre suppressor mutations in Salmonella typhimurium were selected. The amino acid insertions directed by the suppressors were inferred from suppression patterns of Escherichia coli lacI amber mutations. These amber mutations only respond to nonsense suppressors that direct the insertion of particular amino acids. Four Salmonella amber suppressors characterized insert serine, glutamine, tyrosine, and (probably) leucine. Of the three ochre suppressors characterized, two direct the insertion of tyrosine and one directs that of lysine. Of the three amber and two ochre suppressors which have been mapped by phage P22 cotransduction, all are located in the same relative position on the Salmonella map as the analogous E. coli suppressors are on the E. coli map.