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PubMedDate not supplied

Introduction of the emm6 gene into an emm-deleted strain of Streptococcus pyogenes restores its ability to resist phagocytosis.

To complete the formal proof that the M protein functions to protect the group A streptococcus from phagocytosis and to lay the groundwork for the molecular genetic dissection of this major virulence factor, we have reintroduced a wild-type allele of the M protein gene (emm) into the chromosome of a strain from which it had previously been deleted. For this purpose, we used homologous recombination with an engineered plasmid that was introduced into the Streptococcus pyogenes chromosome by an electroporation technique that we developed. The plasmid pJRS120E was constructed in Escherichia coli and contains the structural gene for the antiphagocytic M6 protein (emm6) of S. pyogenes JRS4 and a selectable marker (ermAM) between the chromosomal regions that flank emm6 in S. pyogenes. This plasmid was introduced into S. pyogenes strain JRS75, a strain that is not resistant to phagocytosis as a result of the allelic replacement of the emm6 gene by a kanamycin resistance gene (aphA3). DNA hybridization analysis indicated that in erythromycin-resistant strain JRS115, homologous recombination resulted in the replacement of the aphA3 gene of JRS75 with the introduced emm6 allele. Strain JRS115 produces approximately the same amount of M6 as does the parental emm6+ strain JRS4, as assayed by Western blot analysis, and is resistant to phagocytosis.

Alleles↗
PubMed2001

Role of SspA in the density-dependent expression of the transcriptional activator AarP in Providencia stuartii.

The AarP protein in Providencia stuartii encodes a small transcriptional activator which activates the chromosomal aminoglycoside acetyltransferase aac(2')-Ia gene. In addition, AarP activates genes involved in a multiple antibiotic resistance (Mar) phenotype. Expression of an aarP-lacZ fusion increased in a density-dependent manner and reached peak levels at stationary phase. The expression of an aarP-lacZ fusion could be prematurely activated in cells at early to mid-exponential phase by the addition of spent culture supernatants from stationary phase cultures or by ethyl acetate extracts of these supernatants. Nutrient starvation had a negligible effect on aarP expression. In a search for mutations that block aarP activation at stationary phase, a mini-Tn5Cm insertion has been identified within a gene whose product was 77% identical to SspA, a regulatory protein involved in stationary phase gene expression and virulence. An unmarked sspA null allele (sspA2) was created by allelic replacement to further examine the role of sspA in P. stuartii. The sspA2 allele resulted in substantial decrease in aarP mRNA accumulation at various phases of growth. Furthermore, in an sspA mutant background, the aarP-lacZ fusion was no longer activated by an extracellular signal.

Adhesins, Bacterial↗
PubMed2025

Naturally occurring variation in a cytochrome P450 modifies thiabendazole responses independently of beta-tubulin.

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35D1 and the nuclear hormone receptor nhr-176. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of cyp-35D1 and nhr-176 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive strain, and reciprocal allele replacement strains in different genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele was deleterious, but the lysine allele was selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance.

Animals↗
PubMed1992

Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange.

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.

Alleles↗
PubMed2001

Temporal nucleotide changes in pertactin and pertussis toxin genes in Bordetella pertussis strains isolated from clinical cases in Poland.

Growing number of Bordetella pertussis infections in 1997-1998 in Poland overshadowed the successful national vaccination program. This situation prompted us to investigate if this shift reflects changes in the B. pertussis population. We investigated the possible divergence in genes encoding pertussis toxin subunit 1 (PtxS1) and pertactin (P.69) in B. pertussis population strains during the period of 1960-2000. The pertussis toxin and pertactin variants (ptxS1B and prn1) were found in strains used for production of the whole-cell pertussis vaccine (WCV) production in Poland. Results of the study indicate that the ptxS1A-allele replaced the vaccine variant in 69% in the 1960s, and in 100% in 1990s, and although the prn1-allele was found in all strains from the 1960s and 1970s, after 1995 was gradually replaced by prn2 and prn4 variants. Presumably, vaccination could affect the population structure of B. pertussis in Poland and resulted in antigenic shift in both genes analyzed. Our findings may have implications for the composition of polish WCV and the currently licensed acellular pertussis vaccines.

Alleles↗
PubMed2004

Systematic mutagenesis of the Escherichia coli genome.

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the lambdaRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.

Alleles↗
PubMed1992

UV mutagenesis in Salmonella typhimurium is umuDC dependent despite the presence of samAB.

We investigated the role of the umuDC and samAB operons in the UV mutability of Salmonella typhimurium. umuDC is located on the chromosome, whereas samAB resides on the virulence plasmid pSLT. Using allele replacement and plasmid curing techniques, we found that UV mutability was eliminated when any of three different umuDC alleles (umuD1, umuC1, or umuD1 umuC1) were on the chromosome even when samAB was present. We conclude that samAB normally does not complement umuDC function in S. typhimurium.

Alleles↗
PubMed1976

The evolution of the cooperative group.

A simple model, illustrating the transition from a population of free swimming, solitary cells (Chlamydomonas-type) to one consisting of small colonies (Gonium-type) serves as a basis to discuss the evolution of the cooperative group. The transition is the result of a mutation of the dynamics of cell division, delayed cell separation leads to colonies of four cells. With this mutation cooperative features appear, such as synchronised cell divisions within colonies and coordinated flagellar function which enables the colony to swim in definite directions. The selective advantages under given, environmental conditions are defined and the periods necessary for complete allelic replacement in small populations are calculated for asexual and sexual reproduction. The assumption of a steady-state population during allelic substitution is critically considered, particularly under conditions of competition. It is shown that density-dependent population control must operate in the process of selection, Sexual reproduction slows down the rate of selection even though all cells are haploid. This phenomenon can be explained in general terms of "organizational dominance', where individual units coordinate the function of their neighbours which may be of a different allelotype. Cooperativity is pointed out as an a priori systemic feature which resides in the sub-units of systems, group formation and coordination appears thus as an almost inevitable event. A particular type of system described as 'closed cycle of positive fitness interaction' is discussed in more detail. It has the remarkable feature that its members cannot compete with each other; selection takes place between whole cycles (in analogy to Eigen's 1971 model). Gonium has a wide spectrum of 'somatic plasticity' which enables it to assume various colonial configurations depending on physiological and environmental conditions. This feature can be explained as the result of dynamic flexibilities on the macro-molecular level. The particular relationship between the vast, molecular complexity and the relative simple dynamics of the cell cycle must lead eventually to the genetic fixation of an environmentally induced phenotype.

Animals↗
PubMed1998

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Alleles↗
PubMed1998

Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals No essential role for the protein in the growth and virulence of Mycobacterium intracellulare.

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

Animals↗
PubMed2000

Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis.

Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276). A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.

Amino Acid Sequence↗
PubMed1997

Mutations that render the promoter of the histidine operon of Salmonella typhimurium insensitive to nutrient-rich medium repression and amino acid downshift.

The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.

Amino Acids↗
PubMed2002

Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers.

Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis.

Animals↗
PubMed2003

Fitness effects of DHFR-TS mutations associated with pyrimethamine resistance in apicomplexan parasites.

Pyrimethamine resistance in the malaria parasite Plasmodium falciparum is characterized by specific point mutations in the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously explored the effect of these mutations by engineering homologous alleles of Toxoplasma gondii DHFR-TS, which can readily be expressed as recombinant protein for enzymatic studies, or as allelic replacements in transgenic parasites. In order to directly assess the costs of pyrimethamine-resistance in vivo, we have carried out competition studies between mixtures of T. gondii tachyzoites harbouring wild-type or mutant DHFR-TS alleles, both in tissue culture and in mice. Arg59+Asn108 mutants (using the P. falciparum numbering system) exhibit no significant fitness defects in vitro, but a fitness defect of 1.8% per generation in mice. Arg59+Ser223 mutants exhibit fitness defects of >2.8% per generation both in vitro and in vivo, which may explain why this highly pyrimethamine-resistant allele has not been observed in the field. It is important to note that long-term propagation of parasites in vitro or in vivo can produce adaptations affecting fitness by >3.7% per generation, necessitating careful attention to background in head-to-head competition studies. A sensitive PCR-based assay permits different growth rates to be assessed even in the absence of a drug resistance marker that can be scored by plaque assay.

Animals↗
PubMed1984

Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.

Two temperature-sensitive mutations have been constructed in the single actin gene (ACT1) of the yeast Saccharomyces cerevisiae by in vitro mutagenesis of the cloned gene followed by integrative transformation of mutagenized DNA into yeast cells. A strategy of allele replacement was used that allowed recessive mutations to be phenotypically expressed in the initial transformants, thus simplifying the screening of large numbers of independently transformed cells. After confirming that several ts mutations were located within the actin structural gene by genetic methods, these mutant alleles were cloned, and the altered amino acid residues were defined by DNA sequence analysis. The two unique mutations resulted in substitution of proline-32 with leucine and alanine-58 with threonine. In the course of isolating these mutations, the observation was made that a high proportion of yeast cells transformed with exogenous DNA by the spheroplast method are temperature sensitive for growth because of genetic changes unrelated to the transforming DNA.

Actins↗
PubMed2003

STA10 repression of STA gene expression is caused by a defective activator, flo8, in Saccharomyces cerevisiae.

The expression of STA genes that encode extracellular glucoamylase isozymes is repressed in most laboratory Saccharomyces cerevisiae strains, which are believed to contain an undefined repressor, designated STA10. To identify the regulator involved in STA10 repression, we investigate the FLO8, MSN1, MSS11, STE12, and TEC1 genes. The Deltaflo8 or Deltamss11 deletion mutants in the sta10 genetic background exhibit both a loss of flocculation ability and a reduction in extracellular glucoamylase activity, as in the STA10 strain. Moreover, the STA10 repression is suppressed completely or partially by the introduction of a single copy of the FLO8 or MSS11 genes. Sequence analysis and complementation testing of the STA10 strain reveal that it has an inactive, mutated flo8-1 allele. A random spore analysis and transplacement (allele replacement) experiment confirms that the repressive phenotype of STA10 is due to the amber mutation of the transcriptional activator, FLO8.

Alleles↗
PubMed1997

Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus.

A system for high-efficiency gene replacement in Staphylococcus carnosus and Staphylococcus xylosus has been developed, that is based on temperature-sensitive Escherichia coli-Staphylococcus shuttle vectors for fragment delivery and erythromycin resistance cassettes to facilitate selection of genomic copies of disrupted genes. The approach was tested by constructing a phosphotransferase-deficient mutant of S. carnosus and an S. xylosus mutant strain unable to utilize sucrose. Allelic replacements were observed at rather high frequencies, ranging from approximately 10% for the ptsI gene in S. carnosus up to 50% for the scrB gene in S. xylosus. These differences most likely reflect the length of homology rather than strain-specific variations in recombination efficiencies. Apart from the staphylococcal species tested in this study, the system appears to be applicable in other staphylococci.

Alleles↗
PubMed2005

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A.

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.

Amino Acid Sequence↗