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PubMed1999

Targeted gene disruption reveals an adhesin indispensable for pathogenicity of Blastomyces dermatitidis.

Systemic fungal infections are becoming more common and difficult to treat, yet the pathogenesis of these infectious diseases remains poorly understood. In many cases, pathogenicity can be attributed to the ability of the fungi to adhere to target tissues, but the lack of tractable genetic systems has limited progress in understanding and interfering with the offending fungal products. In Blastomyces dermatitidis, the agent of blastomycosis, a respiratory and disseminated mycosis of people and animals worldwide, expression of the putative adhesin encoded by the WI-1 gene was investigated as a possible virulence factor. DNA-mediated gene transfer was used to disrupt the WI-1 locus by allelic replacement, resulting in impaired binding and entry of yeasts into macrophages, loss of adherence to lung tissue, and abolishment of virulence in mice; each of these properties was fully restored after reconstitution of WI-1 by means of gene transfer. These findings establish the pivotal role of WI-1 in adherence and virulence of B. dermatitidis yeasts. To our knowledge, they offer the first example of a genetically proven virulence determinant among systemic dimorphic fungi, and underscore the value of reverse genetics for studies of pathogenesis in these organisms.

Alleles↗
PubMed2006

Four linked genes participate in controlling sporulation efficiency in budding yeast.

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae. Following the identification of single nucleotide polymorphisms by comparing the sequences of 145 genes between the parental strains SK1 and S288c, we analyzed the segregating progeny of the cross between them. Through reciprocal hemizygosity analysis, four genes, RAS2, PMS1, SWS2, and FKH2, located in a region of 60 kilobases on Chromosome 14, were found to be associated with sporulation efficiency. Three of the four "high" sporulation alleles are derived from the "low" sporulating strain. Two of these sporulation-related genes were verified through allele replacements. For RAS2, the causative variation was suggested to be a single nucleotide difference in the upstream region of the gene. This quantitative trait nucleotide accounts for sporulation variability among a set of ten closely related winery yeast strains. Our results provide a detailed view of genetic complexity in one "QTL region" that controls a quantitative trait and reports a single nucleotide polymorphism-trait association in wild strains. Moreover, these findings have implications on QTL identification in higher eukaryotes.

Alleles↗
PubMed1997

The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase.

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow micro-aerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.

Alleles↗
PubMed1998

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola.

The pore-forming major surface protein (Msp) and the chymotrypsin-like protease (CTLP) of Treponema denticola ATCC 35405 have been implicated in periodontal pathogenicity. Allelic replacement mutations were constructed at two sites in the msp gene and at one site in the CTLP locus. All mutant strains lacked the Msp hexagonal array outer membrane ultrastructure. Strain CKE, in which the locus encoding CTLP was disrupted, produced no CTLP and had aberrant Msp expression. The msp mutant MHE lacked Msp, and produced increased levels of CTLP. In contrast, msp mutant MPE made small amounts of a truncated Msp, but produced no CTLP. Interactions between Msp and CTLP in transport or assembly of the outer membrane complex are proposed.

Alleles↗
PubMed2000

Virulence of a Proteus mirabilis ATF isogenic mutant is not impaired in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a common cause of urinary tract infection, produces a number of different fimbriae, including ambient temperature fimbriae (ATF). These fimbriae are optimally expressed at 23 degrees C and their contribution to urinary tract infection has so far remained unknown. In the present study, a clinical isolate of P. mirabilis and an isogenic allelic replacement mutant unable to express ATF were tested for their ability to cause infection in the ascending urinary tract infection model in mice. The atf mutant colonised the urinary tract as well as the wild-type strain and was also able to outcompete the wild-type strain in a co-challenge experiment. Different non-clinical P. mirabilis isolates showed a reactive AtfA band after Western blot analysis using a polyclonal rabbit AtfA antiserum. These data together suggest that ATF does not play a role in P. mirabilis urinary tract infection.

Alleles↗
PubMed1997

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2).

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.

Alleles↗
PubMed2000

impA, a gene coding for an inner membrane protein, influences colonial morphology of Actinobacillus actinomycetemcomitans.

Directed mutagenesis of a gene coding for a membrane protein of the periodontopathogen Actinobacillus actinomycetemcomitans was achieved by conjugation. The gene was disrupted by insertion of an antibiotic cassette into a unique endonuclease restriction sequence engineered by inverse PCR. The disrupted gene was cloned into a conjugative plasmid and transferred from Escherichia coli to A. actinomycetemcomitans. The allelic replacement mutation resulted in the loss of a 22-kDa inner membrane protein. The loss of this protein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in impA was a change in the pattern of growth of the mutant bacteria in broth cultures. The progenitor bacteria grew as a homogeneous suspension of cells compared to a granular, autoaggregating adherent cell population described for the mutant bacteria. These data suggest that ImpA may play a regulatory role or be directly involved in protein(s) that are exported and associated with colony variations in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter.

A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

Bacterial Adhesion↗
PubMed2004

Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis.

Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. In a previous study, an iglC null mutant of F. tularensis live vaccine strain LVS was generated by allelic replacement and in the current study this iglC mutant was successfully complemented in trans. We characterized the capacity of this iglC mutant and the complemented strain to induce macrophage apoptosis. The iglC mutant did not induce apoptosis in the infected cells. In contrast, the complemented iglC strain was able to multiply in the murine macrophage-like cell line J774A.1 and induced apoptosis similar to that of the wild-type strain. It is the first successful example of complementation in trans of a F. tularensis mutant strain and more importantly this work provides direct evidence that the intracellular growth ability is essential for F. tularensis to induce macrophage apoptosis.

Alleles↗
PubMed2006

A Porphyromonas gingivalis haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion.

Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epithelial cells. In contrast, mutation of a second HAD family serine phosphatase of P. gingivalis (SerB1170), or of a serine transporter, did not affect invasion. A pull-down assay identified GAPDH and heat-shock protein 90 as potential substrates for SerB653. Furthermore, exogenous phosphatase regulated microtubule dynamics in host cells. These data indicate that P. gingivalis has adapted a formerly metabolic enzyme to facilitate entry into host cells by modulating host cytoskeletal architecture. Our findings define a virulence-related role of a HAD family phosphatase and reveal an invasin of an important periodontal pathogen.

Alleles↗
PubMed2003

Geographic variation of the Japanese spinous loach, Cobitis takatsuensis, inferred from allozyme analysis.

Genetic variation among river populations of the Japanese spinous loach, Cobitis takatsuensis, on Shikoku Island, was demonstrated by allozyme electrophoresis based on 40 presumed loci representing 20 enzymes and one non-enzymatic protein. Complete allelic replacement was not evident between populations in any one river system, but existed between populations in different river systems. The maximum genetic distance among populations reached 0.13 and the populations were divided genetically into four or five groups by river system on UPGMA and Neighbor-joining trees. In view of the genetic diversity of C. takatsuensis on Shikoku Island, the populations in three small river systems are thought to have originated from populations in one or the other of two large river systems with a bottle-neck effect. C. takatsuensis is currently designated as an endangered species in Japan. This study suggested that the populations of C. takatsuensis in each river system should be regarded as independent conservation units.

Alleles↗
PubMed1998

Gonococcal invasion of epithelial cells driven by P.IA, a bacterial ion channel with GTP binding properties.

The neisserial porin P.I is a GTP binding protein that forms a voltage-gated channel that translocates into mammalian cell membranes and modulates host cell signaling events. Here, we report that P.I confers invasion of the bacterial pathogen Neisseria gonorrhoeae into Chang epithelial cells and that this event is controlled by GTP, as well as other phosphorus-containing compounds. Bacterial invasion was observed only for strains carrying the P.IA subtype of porin, which is typically associated with the development of disseminated neisserial disease, and did not require opacity outer membrane proteins, previously recognized as gonococcal invasins. Allelic replacement studies showed that bacterial invasiveness cotransferred with the P.IA (por1A) gene. Mutation of the P.I-associated protein Rmp did not alter the invasive properties. Cross-linking of labeled GTP to the porin revealed more efficient GTP binding to the P.IA than P.IB porin subtype. GTP binding was inhibited by an excess of unlabeled GTP, ATP, and GDP, as well as inorganic phosphate, but not by UTP or beta-glycerophosphate, fully in line with the respective invasion-inhibitory activities observed for these compounds. The P.IA-mediated cellular invasion may explain the more invasive behavior of P.IA strains in the natural infection and may broaden the basis for the development of a P.I-based gonococcal vaccine.

Alleles↗
PubMed1994

The identification of rofA, a positive-acting regulatory component of prtF expression: use of an m gamma delta-based shuttle mutagenesis strategy in Streptococcus pyogenes.

Binding of the Gram-positive pathogenic bacterium Streptococcus pyogenes (group A streptococcus) to respiratory epithelium is mediated by the fibronectin-binding adhesin, protein F. Most strains of streptococci regulate the expression of protein F in response to oxygen levels and redox potential; however, JRS4 constitutively binds high levels of fibronectin under all environmental conditions. In this study, we have examined the regulation of protein F expression in JRS4 using a shuttle mutagenesis strategy novel to S. pyogenes. Cloned DNA representing the chromosomal loci adjacent to the gene which encodes protein F (prtF) was subjected to transposon mutagenesis in Escherichia coli using a derivative of transposon m gamma delta that was modified to contain a streptococcal antibiotic-resistance gene. mutagenized DNA was then returned to the streptococcal chromosome by allelic replacement. Analysis of the resulting fibronectin-binding phenotypes revealed that insertions in a region upstream of prtF abolished the constitutive phenotype. However, these mutants now demonstrated regulation in response to both oxygen levels and redox potential. Because these insertions define a locus responsible for the constitutive phenotype, it has been designated rofA (regulator of F). Chromosomal interruption studies using integrational plasmids together with complementation data from a previous study (VanHeyningen et al., 1993) suggested that rofA acts as a positive trans-acting regulator of prtF. Construction of prtF-lacZ fusions indicated that transcription of prtF is constitutive in JRS4 but is regulated in rofA mutants. Analysis of the DNA sequence defined by the rofA insertions revealed a 1495 bp open reading frame, whose predicted product (RofA) possessed both a putative helix-turn-helix motif and limited homology to two other transcriptional activators (Mry, PrgR) of Gram-positive surface proteins. Sequences homologous to rofA were found in regulated strains of S. pyogenes, which suggests that rofA may act as an activator of prtF in response to an unidentified environmental signal. We speculate that the allele reported here contains a mutation that renders it constitutively active.

Amino Acid Sequence↗
PubMed2002

Actinobacillus pleuropneumoniae serotype 1 carrying the defined aroA mutation is fully avirulent in the pig.

The aroA gene from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 was isolated and sequenced. The gene complemented the aroA mutation in Escherichia coli AB2829. A kanamycin resistance cassette was inserted into the aroA gene and the mutant gene was reintroduced into A. pleuropneumoniae by allelic replacement. Intratracheal infection of susceptible pigs with A. pleuropneumoniae aroA caused no signs of respiratory disease or lung lesions in any of the animals at a dose 10(4) times the dose reliably known to induce acute pleuropneumonia; all animals infected with the unaltered control strain developed acute disease. The aroA mutant was rapidly eliminated from the lungs and tonsil of infected animals. The mutant may represent a safely attenuated strain for use in live bacterial vaccination or the delivery of antigen by the intranasal route. However, the residence time of the mutant in the respiratory tract of the pig may be too short for it to be useful in generating a protective immune response.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1990

Mutations that affect transcription and cyclic AMP-CRP regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.

We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.

Acetates↗
PubMed2000

Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria.

Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

Alkyl and Aryl Transferases↗
PubMed1996

Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureus decreases virulence in experimental wound infection.

A mutant deficient for the 19-kDa extracellular fibrinogen-binding protein (Fib) from Staphylococcus aureus has been constructed. The gene was inactivated by allele replacement. A 2.0-kb fragment from transposon Tn4001 carrying the gene for gentamicin resistance was inserted into the gene encoding Fib (fib). The genotype was verified by PCR analysis, and the loss of Fib was demonstrated by Western blotting (immunoblotting). The mutation has not altered the ability of the strain to bind to fibrinogen or fibronectin compared with that of the isogenic parental strain, FDA486. The mutant, designated K4.3, was compared with strain FDA486 in a wound infection model in rats. Sixty-eight percent of the rats challenged with parental strain FDA486 developed severe clinical signs of wound infection, whereas only 29% of the animals challenged with isogenic mutant K4.3 showed severe symptoms (P < 0.01). The weight loss of animals infected with the wild type was also significantly different from that of animals infected with the mutant strain. The result demonstrates that the extracellular 19-kDa fibrinogen-binding protein from S. aureus contributes to the virulence in wound infection and delays the healing process.

Alleles↗
PubMed1998

Role of the Haemophilus ducreyi Ton system in internalization of heme from hemoglobin.

By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressing hgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes was exbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.

Aldehyde Oxidoreductases↗