Contraselectable streptomycin susceptibility determinant for genetic manipulation and analysis of Helicobacter pylori

Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str(r) allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str(r)] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm(r)] and Str(s)) were each transformed with wild-type genomic DNAs, and Str(r) derivatives were selected. The desired Erm(s) Str(r) isolates were obtained at frequencies that ranged from 17 to 96% among Str(r) transformants, with the Erm(s) yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.     

Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str(r)), gentamicin, or rifampin (Rif(r)). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str(r) mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif(r) mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase beta subunit). Increased productivity of salinomycin in the Str(r) mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg(2+) concentrations. We conclude that this aberrant protein synthesis ability in the Str(r) mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

Isolation and characterization of sporulation-initiation mutation in the Bacillus subtilis prfB gene

A Bacillus subtilis prfB45 mutant grew at 42 degrees C, but its sporulation was severely defective at 37 degrees C. Sporulation-specific induction of kinA, spo0A, and spo0H genes was inhibited in the mutant. The effects of temperature up-shift and down-shift on sporulation of the prfB45 mutant was observed at an early stage of sporulation. UGA readthrough frequency at non-permissive temperatures for sporulation was higher in the mutant than in the wild-type strain. Temperature-sensitive sporulation of the prfB45 mutant was suppressed by mutations in rpsL coding for S12 of ribosomes, required for accurate termination of translation. Additionally, spontaneous second-site mutations that suppressed the sporulation phenotype of the prfB45 strain were found in the rpoB gene. These results suggest that accurate termination of translation is required for proper initiation of sporulation.     

Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

Comparative analysis of UV-induced mutability of ten different codon units in position 211 of the Escherichia coli trpA gene

Summary To study the influence of the local base, composition upon UV-induced mutability the reversion frequencies of ten trpA mutants with known codon sequences at position 211 were compared. Comparison of mutant strains reverting by the same base substitution type but with different dipyrimidinic, sequences reveals the mutagenic character of the pyrimidine-pyrimidine (6-4) photoproduct. The codons GAC and GAT, both reverting by AT-GC transitions and AT-CG transversions in the middle position and harboring the dipyrimidinic sequence CT in the opposite strand, differ, in their reversion frequencies sixfold. This difference can only be due to the influence of the different bases in the third codon position upon the mutability of adjacent second bases.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.     

Suppressors of lysine codons may be misacylated lysine tRNAs

We describe a novel class of missense suppressors that read the codons for lysine at two positions (211 and 234) in the trpA polypeptide of Escherichia coli. The suppressor mutations are highly linked to lysT, a gene for lysine tRNA. The results suggest that the suppressors are misacylated lysine tRNAs that carry glycine or alanine. The mutant codons are apparently suppressed better at position 211 than at position 234, indicating the existence of codon context effects in missense suppression.     

Hypermutability of a UV-sensitive aphidicolin-resistant mutant of Chinese hamster fibroblasts

An ultraviolet light (UV)-sensitive thymidine auxotroph of Chinese hamster V79 cells that exhibits pleiotropic effects such as a high level of deoxycytidine triphosphate, slow growth, sensitivity to cytidine, and high frequencies of site-specific bromodeoxyuridine-dependent chromosomal aberrations was selected by its resistance to aphidicolin. The UV-induced mutability of this mutant and one of its revertants, which retains some of the phenotypes listed above, was studied in 3 mutation assay systems. The results showed that the mutant was hypermutable for ouabain and diphtheria-toxin-resistant mutations compared to wild-type V79 cells at the same UV dose or the same survival level. The mutant exhibits a delayed expression of maximal frequency of induced 6-thioguanine-resistant mutants. When maximal frequencies are compared at the same UV dose, the mutant also has higher mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase locus. The revertant was similar to the wild-type in UV sensitivity and mutability. Our results showed that UV sensitivity and hypermutability are correlated and controlled by a single gene. Thymidine auxotrophy or nucleotide pool imbalance apparently did not contribute to the UV sensitivity and mutability of the mutant.     

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.     

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Development and use of a gene deletion strategy for Flavobacterium johnsoniae to identify the redundant gliding motility genes remF, remG, remH, and remI

Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmid restored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type rpsL and used streptomycin for counterselection. This approach was used to delete the region spanning remF, remG, and fjoh_0982. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had no effect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The rpsL counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of F. johnsoniae physiology.     

MNNG-induced mutations in the adult gill and hepatopancreas and in embryos of rpsL transgenic zebrafish

To evaluate the feasibility of a mutagenicity assay using adult rpsL transgenic zebrafish, 4- to 8-month-old females were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0, 15 or 30 mg/L in a water bath for 2 h). At 2 weeks after exposure, MNNG showed a concentration-dependent significant increase in mutant frequency (MF) of 8 x 10(-5), 18 x 10(-5), and 51 x 10(-5), respectively, in the gill. DNA sequencing revealed that 60-74% of the induced mutations were G:C to A:T transitions, consistent with the known mutagenic effects of MNNG. A marginal but significant increase in MF was observed in the hepatopancreas only in the group exposed to 30 mg/L, with the induction of some G:C to A:T transitions. A time-course of the appearance of mutations was determined in fish treated with 15 mg/L MNNG. In both, the gill and hepatopancreas, a higher MF was observed at 3 weeks than at 2 weeks, suggesting that an expression time of at least 3 weeks is preferable for the assay. When embryos (29 h post-fertilization) were exposed to MNNG (0, 50, and 150 mg/L) for 1 h, MFs increased significantly with an increase in the concentration of MNNG (5 x 10(-5), 40 x 10(-5), and 144 x 10(-5), respectively) at 3 days after exposure. G:C to A:T transitions were the predominant mutations, and these occurred at the same sites in the rpsL gene as in adult tissues. Thus, MNNG induces typical mutations in the gill and hepatopancreas of adult fish, and in embryos, suggesting that the rpsL zebrafish is a useful tool for monitoring genotoxicity caused by water-borne mutagens.     

Cloning and sequence analysis of the rpsL and rpsG genes of Mycobacterium smegmatis and characterization of mutations causing resistance to streptomycin.

The Mycobacterium smegmatis rpsL and rpsG genes, encoding the ribosomal proteins S12 and S7, were cloned, and their DNA sequence was determined. The third nucleotide of the S12 termination codon overlapped the first nucleotide of the S7 translation initiation codon. A collection of 28 spontaneous streptomycin-resistant mutants of M. smegmatis were isolated. All had single-base-pair substitutions in the rpsL gene which were changed to a streptomycin-sensitive phenotype by complementation with a low-copy-number plasmid carrying the wild-type M. smegmatis rpsL gene. A total of eight different mutations were found in two specific regions of the rpsL gene. Fifty-seven percent (16 of 28) altered the Lys codon at position 43. Forty-six percent of the mutations (13 of 28) were due to a transition changing an AAG Lys codon to an AGG Arg codon, with eight changes at codon 43 and five at codon 88.     

Identification of a novel mutation affecting domain V of the 23S rRNA gene in Helicobacter pylori

BACKGROUND: This study analyzes clarithromycin resistance status and 23S rRNA gene mutations in Helicobacter pylori strains from Central Italian patients. MATERIALS AND METHODS: H. pylori strains from 235 dyspeptic patients (205 with no history of clarithromycin exposure and 30 referred for failure of eradication therapy) were tested for clarithromycin resistance by screening agar method and E-test. Resistant strains were analyzed for mutations of the 23S rRNA gene by PCR-RFLP and sequencing. RESULTS: Primary resistance was observed in strains from 43/205 (21%) patients with no history of clarithromycin exposure and secondary resistance in 30/30 (100%) strains from previously treated patients. A single mutant strain was detected in 54/73 (74%) cases, a mixture of one or more mutant(s) plus the wild type in the remaining 19/73 (26%) cases. One 23S rRNA gene mutation (A-->T transversion at nucleotide 2144) in the peptidyltransferase region of domain V was novel. CONCLUSIONS: This study shows: (a) a high prevalence of H. pylori strains with primary or secondary clarithromycin resistance in an urban area of Central Italy; (b) colonization by both mutant and wild-type H. pylori in the same patient; (c) a novel variant of the H. pylori 23S rRNA gene.     

Site Specificity and Variability in the Mutator and Antimutator Effects of Phage T4 Gene 43 Mutants

Spontaneous, 2-aminopurine- and 5-bromouracil-induced mutations at six rII nonsense codons were studied in phage T4 strains possessing wild-type and mutant gene 43 alleles. The mutation pathways studied included interconversions and reversions of nonsense codons. The tsCB87 allele, which specifies an antimutator DNA polymerase, reduced base-analogue-induced mutation frequencies along all pathways. However, GC base pairs were less affected than AT base pairs. The frequency of spontaneous UAA→UAG conversions was also reduced by tsCB87, but that of spontaneous UAA→UGA conversions was often increased. Mutation in the presence of the mutator allele tsL56 was increased along all pathways, with no preference for either AT or GC base pairs. Mutation frequencies in the presence of the two mutant DNA polymerases were highly variable. A strong correlation was found between 2-aminopurine-induced mutation frequencies in ts⁺ and tsCB87 phage along the reversion and UAA→UAG (but not UAA→UGA) pathways.