Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. II. Stimulation of duplication-loss from merodiploids.

Strains of Salmonella typhimurium which contain a duplication of approximately 30% of the genome may be obtained by a simple selective procedure. These strains are highly unstable, losing the duplication when grown on non-selective medium. In this paper we report that treatment of merodiploid bacteria with mutagenic agents stimulates the rate at which haploid segregants are obtained from merodiploid strains. The mutagens which have been tested for this effect are X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), and the azaacridine half-mustard ICR-372.     

Regulation of the hut operons of Salmonella typhimurium and Klebsiella aerogenes by the heterologous hut repressors.

In merodiploid strains of Klebsiella aerogenes with chromosomal hut genes of K. aerogenes and episomal hut genes of Salmonella typhimurium, the repressor of either species can regulate the hut operons of the other species. The repression exerted by the homologous repressor on the left-hand hut operon is, in both organisms, stronger than that exerted by the heterologous repressor.     

Isolation of a trans-dominant histidase-negative mutant of Salmonella typhimurium

A mutation of Salmonella typhimurium was obtained that results in the failure of cells to synthesize the enzyme l-histidine ammonia-lyase (histidase). The mutation mapped within the hutH gene and in merodiploid strains was dominant over the wild-type allele. Extracts from cells bearing the trans-dominant histidase-negative allele were shown to contain material that reacts immunologically with antiserum against purified wild-type histidase. It is proposed that the trans-dominant allele results in the synthesis of defective histidase subunits that can combine with, and partially inactivate, wild-type histidase subunits. This subunit mixing presumably does occur, as the enzyme synthesized in a hybrid merodiploid strain is abnormally heat sensitive.     

Arginine regulon control in a Salmonella typhimurium--Escherichia coli hybrid merodiploid.

Summary The regulation of synthesis of arg enzymes was studied in a hybrid merodiploid in which an episome of Escherichia coli carrying the argR ⁺ allele was transferred to a Salmonella typhimurium argR strain. The arg enzyme levels of the hybrid merodiploid were compared to that found in argR and argR ⁺ haploids of S. typhimurium. The results showed that repression of synthesis of arg enzymes was effected through the introduction of the E. coli argR ⁺ allele but significant quantitative differences of arg enzyme levels in the argR ⁺ haploid and the hybrid merodiploid were observed.     

Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.     

鼠伤寒沙门氏菌嘌呤生物合成基因表达的调控研究——Ⅱ.O~c突变体的分离和遗传鉴定

已有研究证明,编码阻遏蛋白的调节基因purR能调节嘌呤从头合成途径中除purB外所有结构基因的表达。但迄今还缺乏阻遏蛋白与这些基因的操纵基因相结合的直接证据。本文报道以嘌呤结构基因purD和purG的MudJ(lacZ,Kan~5)插入物为出发株,在外加过量腺嘌呤核苷(2mmol/L)的MacConkey平板上通过选择红色菌落分离O~c突变体的结果。从上述两株出发株分别获得了8株和9株独立的消阻遏突变体。共转导分析和顺反试验证明,两组突变体中各有1株顺式作用突变体(O~c)。这是在鼠伤寒沙门氏菌中首次获得的嘌呤O~c突变体,为研究阻遏蛋白与操纵基因相互作用提供了重要材料。     

Salmonella typhimurium newD and Escherichia coli leuC genes code for a functional isopropylmalate isomerase in Salmonella typhimurium-Escherichia coli hybrids.

The supQ newD gene substitution system in Salmonella typhimurium restores leucine prototrophy to leuD mutants by providing the newD gene product which is capable of replacing the missing leuD polypeptide in the isopropylmalate isomerase, a complex of the leuC and leuD gene product. Mutations in the supQ gene are required to make the newD protein available. An Escherichia coli F' factor was constructed which carried supQ- newD+ from S. typhimurium on a P22-specialized transducing genome. This F' pro lac (P22dsupQ394newD) episome was transferred into S. typhimurium strains containing th leuD798-ara deletion; the resulting merodiploid strains had a Leu+ phenotype, indicating that supQ- newD+ is dominant over supQ+ newD+, and eliminating the possibility that the supQ gene codes for a repressor of the newD gene. Furthermore, transfer of the F' pro lac (P22dsupQ39newD) into E. coli leuD deletion strains restored leucine prototrophy, showing that the S. typhimurium newD gene can complment the E. coli leuC gene. Growth rates of the S. typhimurium-E coli hybrid strains indicated that the mutant isopropylmalate isomerase in these strains does not induce a leucine limitation, as it does in S. typhimurium leuD supQ mutants. In vitro activity of the mutant isopropylmalate isomerase was demonstrated; the Km values for alpha-isopropylmalate of both the S. typhimurium leuC-newD isomerase and the S. typhimurium-E. coli hybrid isomerase were as much as 100 times higher than the Km values for alpha-isopropylmalate of the wild-type enzyme, which was 3 x 10(-4) M. Mutagenesis of E. coli leuD deletion strains failed to restore leucine prototrophy, indicating that E. coli does not have genes analogous to the S. typhimurium supQ newD genes, of that, if present, activation of a newD is a rare event or is lethal to the cell.     

Accessory replicons of species of Salmonella and Shigella.

Shigella and Salmonella strains isolated from clinical samples were examined. Out of 42 Shigella strains tested, 17 (40%) were found to be colicinogenic and another 3 were lysogenic. All three lysogens yielded a phage antigenically homologous to coliphage P2. Out of 30 strains tested, only 1 was found to be resistant to both neomycin and sulfamethoxazole. Out of 48 strains of Salmonella tested for drug resistance, only 2 showed multiple drug resistance. In contrast to Shigella isolates, the Salmonella isolates were infrequently (approximately 5%) bacteriocinogenic. The frequency of lysogeny in Salmonella strains was found to be 6% when tested on Salmonella typhimurium LT2, but by using a set of five indicators belonging to species Salmonella potsdam, Salmonella mbadanka, Salmonella dublin, Salmonella london, and Salmonella wandsworth, 50% of the strains were shown to be lysogenic. Salmonella phages related to P22 were recoverable from Salmonella saintpaul, Salmonella indiana, and Salmonella heidelberg. Some isolates of S. typhimurium yielded a temperature-sensitive and P22-heterologous phage which was found to be a more efficient transducer of bacterial genetic markers than P22. EcoRI-generated fragments of the DNA of some phages permitted the establishment of a clonal descent for some of the wild-type lysogenic bacterial strains. This last observation points out the potential usefulness of prophages as epidemiological markers.     

Genetic duplications in bacteria and their relevance for genetic toxicology

Tandem genetic duplications of various lengths occur at high frequency and at many chromosomal locations in bacteria. Most duplications are formed and lost by recombinational mechanisms. Since they readily give rise to haploid segregants, duplications are characteristically unstable. Various selection procedures permit measurements of duplication frequencies, and several mutagens have been shown to induce the formation of duplications in haploid and the loss of duplications from merodiploid bacteria. Although the data base is not extensive, it includes agents that interact with DNA by a variety of molecular mechanisms. Grounds on which the induction of genetic duplications in bacteria can be relevant for genetic toxicology are discussed.     

Frequency of IS1-mediated molecular events in different members of the family Enterobacteriaceae.

The numbers of chromosomal copies of the insertion sequence IS1 in strains of Salmonella typhimurium (0 to 8 copies), Shigella sonnei (56 copies), and Shigella flexneri (41 copies) isolated in Mexico City, Mexico, were similar to those reported for these genera isolated in other countries. Of the 11 Shigella strains studied, all carried several small plasmids; however, in only one of these strains did a small plasmid contain IS1, IS1 recombination, cointegrate formation mediated by IS1 or by the IS1-flanked transposon Tn9, and transposition of Tn9 occurred at a higher frequency in S. typhimurium than in either Escherichia coli or S. sonnei strains. The frequencies of IS1 recombination in S. typhimurium strains containing either zero or eight copies of IS1 were similar.     

Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-pressure liquid chromatography.

A method is described whereby the presence of less than five salmonellae was detected per milliliter of milk within 24 h of sample collection. Salmonellae were removed from milk by means of electropositive large-pore filters. Eluates from the filters were analyzed for the presence of Salmonella spp. by Felix-O1 bacteriophage and high-pressure liquid chromatographic techniques. The method gave only a positive response when salmonellae were present in the milk. Of the serotypes and strains of Salmonella spp. tested, Salmonella dublin (10 strains), Salmonella typhimurium (5 strains), Salmonella anatum, Salmonella krefeld, and Salmonella saint-paul gave positive responses. One strain of Salmonella agona (three strains tested) and three strains of Salmonella enteritidis (seven strains tested) were not detectable by the method described herein.     

Effect of hyperbaric oxygenation on the survivability, mutagenesis and recombinogenesis of single-cell organisms

The hyperbaric oxygenation (HBO) has been studied for its ability to induce the cell death and different genetic variations in normal and repair defective strains of bacteria (Escherichia coli and Salmonella typhimurium) and of yeast (Saccharomyces cerevisiae) strains with different ploidy. HBO is shown to induce an increase in the mutability of excision-repair defective strains and a decrease in the survival rate of error-prone repair defective strains of the procaryote. HBO is a weak recombinogen for yeast and induces no mutations of the haploid strain. The results obtained reflect doubt on the community of the mechanisms of radiation and HBO actions on the cell.     

Inactivation of mismatch repair overcomes the barrier to transduction between Salmonella typhimurium and Salmonella typhi.

P22 transduction of chromosomal genes from Salmonella typhimurium into Salmonella typhi occurs at a low frequency. Transduction of plasmids from S. typhimurium into S. typhi occurs at a frequency similar to that between S. typhimurium strains, indicating that the barrier to transduction of chromosomal genes is not due to an inability of P22 to inject DNA into S. typhi or a restriction endonuclease that rapidly degrades foreign DNA. Furthermore, transduction of mutS and mutL derivatives of S. typhi with chromosomal genes from S. typhimurium occurs efficiently. These results indicate that the transduction barrier is due to activity of the recipient mismatch repair system, which senses sequence divergence and disrupts heteroduplexes in favor of recipient sequences. Inactivation of the mismatch repair system allows P22 transduction to be used as an effective tool for constructing S. typhi-S. typhimurium hybrids.     

Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-pressure liquid chromatography

A method is described whereby the presence of less than five salmonellae was detected per milliliter of milk within 24 h of sample collection. Salmonellae were removed from milk by means of electropositive large-pore filters. Eluates from the filters were analyzed for the presence of Salmonella spp. by Felix-O1 bacteriophage and high-pressure liquid chromatographic techniques. The method gave only a positive response when salmonellae were present in the milk. Of the serotypes and strains of Salmonella spp. tested, Salmonella dublin (10 strains), Salmonella typhimurium (5 strains), Salmonella anatum, Salmonella krefeld, and Salmonella saint-paul gave positive responses. One strain of Salmonella agona (three strains tested) and three strains of Salmonella enteritidis (seven strains tested) were not detectable by the method described herein.     

Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

SALMONELLA TYPHIMURIUM-ESCHERICHIA COLI Hybrids for the Tryptophan Region

Fifty-eight hybrids were analyzed for their phenotypic stability, presence and nature of cryptic trp alleles and by P22-mediated transduction to yield percent homologies. The hybrids fall into 5 distinguishable classes: a haploid class in which selected E. coli genes replace equivalent sites in the S. typhimurium chromosome; three merodiploid classes in which the selected E. coli genes are integrated at novel sites in the S. typhimurium chromosome-on the same transducing fragment as the female genes selected against, with or without cryptic damage to a nearby gene, or not on the same transducing fragment; and one class in which recombination has not taken place and the E. coli DNA is presumed to be an exogenote. The homology values are heterogeneous and do not permit an accurate determination of the relative frequency of incorporation of the integrated male genetic material. A further study of 20 hybrids indicates that genetic rearrangements can occur in the hybrids.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Involvement of ribosomal ribonucleic acid operons in Salmonella typhimurium chromosomal rearrangements.

As part of our efforts to understand factors influencing chromosomal organization and rearrangements, we studied a family of Salmonella typhimurium tandum duplication mutants. We found that the duplications were originally generated by unequal recombination between pairs of similarly oriented ribosomal ribonucleic acid operons (rrn). This demonstration involved the physical isolation of the duplicated material as circular deoxyribonucleic acid excised from the duplication. The four rrn operons involved embraced the ilv pur D segment of the chromosome and occurred at positions closely analogous to those previously observed for Escherichia coli. The interval between rrnC and rrnA of S. typhimurium was similar in size to that of E. coli (43 versus 39 kilobases), as was the interval between rrnB and rrnE (94 versus 91 kilobases). The rrnA-to-rrnB interval of S. typhimurium, however, was 155 kilobases, substantially greater than the 126 kilobases observed for E. coli.     

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.     

Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.