Salmonella typhimurium hisG46 (R-Utrecht): Possible Use in Screening Mutagens and Carcinogens

Salmonella typhimurium LT2 hisG46 becomes a more sensitive strain for assaying mutagens and carcinogens when it carries the resistance transfer factor R-Utrecht.     

On the different response of Salmonella typhimurium hisG46 and TA1530 to mutagenic action of base analogues.

Various mutagens are known to induce more his+ revertants in TA1530 than in hisG46 strain. To test whether the mutator effect shown by TA1530 is limited to the his mutation, the lysA8 marker was introduced into both the TA1530 and hisG46 strain, and its reversibility, after induction by N4-hydroxycytidine, was estimated. The ability to reverse the lys marker was tenfold higher in the TA1530/lysA8 transductants than in the hisG46/lysA8 transductants or in the donor for lys, the lysA8 strain.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Phenotypic and reversion analysis of a Salmonella typhimurium constructed to have an arginine codon at the hisG46 missense codon

Of the 6 single-base mutations that would be predicted to change the missense mutation hisG46 away from a proline codon in the Salmonella/microsome mutagen selection assay for histidine-independent revertants, only 5 have been observed. We have used site-specific mutagenesis to make the unobserved mutant [CCC (proline)----CGC (arginine)] codon in the Salmonella genome. Experiments with this arginine mutant demonstrate that, like bacteria containing the hisG46 mutation, bacteria with the arginine missense mutation are histidine auxotrophs which are capable of reversion to histidine independence. However, unlike the ATP phosphoribosyltransferase coded by the hisG46 his G gene (with a proline), the arginine mutant enzyme is partially active. This is indicated by a histidine-independent phenotype when the arginine hisG gene is present in multiple copies.     

Influence of the processing of MucA protein on the reversion of the frameshift hisD3052 and the base substitution hisG46 mutations by chemical mutagens in Salmonella typhimurium

The correlation between the proficiency at promoting mutagenesis of MucA/B proteins and MucA processing has been considered to be very high (Hauser et al., 1992) on the basis of the results of UV mutagenicity (Shiba et al., 1990). Here we show that this correlation is only partial. We have assayed the mutagenicity of benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1) in Salmonella typhimurium tester strains containing plasmids which encode MucA proteins with an altered cleavage site. Reversion of the frameshift hisD3052 mutation by B[a]P or AFB1 was observed in the presence of non-cleavable MucA protein although at a lower level than that found in cells containing wild-type MucA protein. Reversion of the base substitution hisG46 mutation by AFB1 requires a significant processing of MucA, while lower levels of this processing would be enough for the hisG46 reversion by B[a]P. These results suggest that the specificity of mutations induced by mutagens forming DNA adducts is influenced by the activity of MucA protein. They also show the relevance of mutagenicity assays in the mechanistic studies of mutagenesis.     

Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences.

The fliG, fliM, and fliN genes of Salmonella typhimurium encode flagellar components that participate in energy transduction and switching. We have cloned these genes and determined their sequences. The deduced amino acid sequences correspond to proteins with molecular masses of 36,809, 37,815, and 14,772 daltons, respectively. None of the protein sequences are especially hydrophobic or look as though they correspond to integral membrane proteins, a result consistent with other evidence suggesting that the proteins may be peripheral to the membrane, possibly mounted onto the basal body M ring. The fliL gene, which immediately precedes fliM, is of unknown function; it encodes a protein with a deduced molecular mass of 17,082 daltons. The hydropathy profile of FliL indicates that it is likely to be an integral membrane protein with at least one spanning segment, near its N terminus. None of the four proteins exhibit consensus N-terminal signal sequences. Comparison of the fliL, fliM, and fliN sequences with the homologous ones in Escherichia coli reveals ranges of similarities of 77 to 95% at the amino acid level and 75 to 86% at the nucleotide level, with the majority (58 to 89%) of codon changes being synonymous ones.     

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.     

DNA sequence analysis of revertants of the hisD3052 allele of Salmonella typhimurium TA98 using the polymerase chain reaction and direct sequencing: application to 1-nitropyrene-induced revertants

We have used the polymerase chain reaction (PCR) to speed the DNA sequence analysis of revertants of Salmonella typhimurium TA98. Briefly, a crude DNA extract from a single colony was prepared and used in an asymmetric PCR to amplify a 328-bp fragment containing the hisD3052 mutation approximately in the center. Following ultrafiltration, the ssDNA was sequenced using an end-labeled probe and dideoxy sequencing. The most frequent mutation among the revertants was a -2 deletion of GC or CG within the sequence CGCGCGCG, which is upstream of the hisD3052 mutation. This deletion occurred in 38% (6/16) of the spontaneous (-S9) revertants and in 94% (15/16) of a set of 1-nitropyrene-induced revertants. Other mutations, mostly deletions but also some complex mutations (i.e., single mutational events involving a combination of insertions, deletions, and substitutions), occurred within quasipalindromic regions of DNA. Possible mutational mechanisms are discussed, and the results with 1-NP are compared to those obtained in other systems.     

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes.

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.     

Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation

Antibiotic resistance in bacteria is generally associated with fitness costs that often can be reduced by second-site compensatory mutations. Here, we examined how a protamine-resistant small colony variant of Salmonella typhimurium adapts to the growth reduction conferred by a resistance mutation in hemC (encoding a haem-biosynthesis enzyme). We show that adaptation occurs in a multi-step process where fitness is successively increased. Thus, the initial adaptive response was selection for an unstable gene amplification of the mutant hemC gene that provided a small fitness increase. Fitness was increased further by a mutation that restored HemC function in one gene copy, relaxing selection for the amplification. Subsequently, the amplification segregated back to the haploid state and even higher fitness. The end result was in most cases mutant strains with a hemC sequence different from that of the wild-type strain. These findings suggest that gene amplification facilitates adaptive evolution. A higher gene dosage increases the target size for compensatory mutations and improves fitness of the cell, thereby allowing an increase in the population size, further increasing the probability of a subsequent stable mutation. Our results provide a novel genetic basis for growth compensation in small colony variants.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.     

Hybridization properties of long nucleic acid probes for detection of variable target sequences,and development of a hybridization prediction algorithm

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

Genomic comparison of Salmonella enterica serovars and Salmonella bongori by use of an S. enterica serovar typhimurium DNA microarray

The genus Salmonella consists of over 2,200 serovars that differ in their host range and ability to cause disease despite their close genetic relatedness. The genetic factors that influence each serovar's level of host adaptation, how they evolved or were acquired, their influence on the evolution of each serovar, and the phylogenic relationships between the serovars are of great interest as they provide insight into the mechanisms behind these differences in host range and disease progression. We have used an Salmonella enterica serovar Typhimurium spotted DNA microarray to perform genomic hybridizations of various serovars and strains of both S. enterica (subspecies I and IIIa) and Salmonella bongori to gain insight into the genetic organization of the serovars. Our results are generally consistent with previously published DNA association and multilocus enzyme electrophoresis data. Our findings also reveal novel information. We observe a more distant relationship of serovar Arizona (subspecies IIIa) from the subspecies I serovars than previously measured. We also observe variability in the Arizona SPI-2 pathogenicity island, indicating that it has evolved in a manner distinct from the other serovars. In addition, we identify shared genetic features of S. enterica serovars Typhi, Paratyphi A, and Sendai that parallel their unique ability to cause enteric fever in humans. Therefore, whereas the taxonomic organization of Salmonella into serogroups provides a good first approximation of genetic relatedness, we show that it does not account for genomic changes that contribute to a serovar's degree of host adaptation.     

Analysis of brevibacteria on the surface of Gruyère cheese detected by in situ hybridization and by colony hybridization

From the surface microflora of three different mature Gruyère Suisse cheeses, only a minor part of the microscopically visible resident micro-organisms was culturable. This may be due to the selectivity of the medium and growth conditions used, but also to dead or dormant cells. However, by use of fluorescent in situ hybridization (FISH) and the bacterial probe EUB338, a major part (70% of 4'6-diamidino-2-phenylindole-stained cells) of the microbial community was detectable. The analysis revealed that, with the aid of further specific nucleotide probes, most of the detectable micro-organisms were attributable to the Actinobacteria (76–90·3%), with comparable results obtained by colony hybridization (82–88%). The percentage of culturable brevibacteria detected by colony hybridization was higher (15–35%) than the percentage determined by in situ hybridization (10·6–14·3%), indicating that the numbers of brevibacteria are overestimated by the plating technique.