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At least 127 records · Page 7Linked to original sources
PubMed2006

M.SpyI, a DNA methyltransferase encoded on a mefA chimeric element, modifies the genome of Streptococcus pyogenes.

While screening the clonality of Streptococcus pyogenes isolates from an outbreak of erythromycin-resistant pharyngitis in Pittsburgh, PA, we found a correlation between the presence of the chimeric element Phi10394.4 (carrying the macrolide efflux gene, mefA) and genomic DNA being resistant to cleavage by SmaI restriction endonuclease. A search of the open reading frames in Phi10394.4 identified a putative type II restriction-modification (R-M) cassette containing a cytosine methyltransferase gene (spyIM). Heterologous expression of the cloned spyIM gene, as well as allelic-replacement experiments, showed that the action of this methyltransferase (M.SpyI) was responsible for the inhibition of SmaI digestion of genomic DNA in the Phi10394.4-containing isolates. Analysis of the methylation patterns of streptococcal genomic DNA from spyIM-positive strains, a spyIM deletion mutant, and a spyIM-negative strain determined that M.SpyI specifically recognized and methylated the DNA sequence to generate 5'-C(m)CNGG. To our knowledge, this is the first methyltransferase gene from S. pyogenes to be cloned and to have its activity characterized. These results reveal why pulsed field gel electrophoresis analysis of SmaI-digested genomic DNA cannot be used to analyze the clonality of some streptococci containing Phi10394.4 and may explain the inability of previous epidemiological studies to use SmaI to analyze DNAs from macrolide-resistant streptococci. The presence of the SpyI R-M cassette in Phi10394.4 could impart a selective advantage to host strain survival and may provide another explanation for the observed increase in macrolide-resistant streptococci.

Alleles↗
PubMed1992

Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker.

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1-type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter-selectable marker, and a chloramphenicol-resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid-borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1-type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose-containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single- or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild-type strain PAO1 and should allow the exchange of any DNA segment into any non-essential regions of the P. aeruginosa chromosome.

Alleles↗
PubMed2003

The Delta subunit of RNA polymerase is required for virulence of Streptococcus agalactiae.

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the United States despite the chemoprophylaxis strategies for preventing infection recommended by the Centers for Disease Control and Prevention. Using signature-tagged transposon mutagenesis to screen for novel virulence factors, we identified the rpoE gene as essential for development of sepsis in a neonatal rat model of GBS infection. An rpoE allelic replacement mutant displayed attenuated virulence in the sepsis model of infection identical to that of the transposon mutant, confirming linkage of the phenotype to the mutation in rpoE. The rpoE mutants also displayed increased sensitivity to killing in whole-blood bactericidal assays, which may explain the attenuated virulence. The mutants were otherwise phenotypically identical to the wild-type strain, including growth rate in plasma, indicating that a growth defect is not responsible for the attenuated virulence. rpoE is found only in gram-positive bacterial species and encodes the delta peptide, a subunit of RNA polymerase. Previous in vitro studies in other bacteria suggest that the delta peptide plays a role in maintaining transcriptional fidelity by blocking RNA polymerase binding at all but the strongest promoters, thereby inhibiting initiation of transcription. Despite the availability of rpoE mutants for several gram-positive bacterial species, a role for the peptide in vivo has not been defined, though it has been postulated that the delta peptide may be important for long-term survival in vitro or during growth phase transitions. Our data represent the first report of a phenotype relevant to virulence for rpoE mutants.

Alleles↗
PubMed2001

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis.

Two-component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two-component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock-out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene-regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium-regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

Alleles↗
PubMed1996

Defined mutants of Proteus mirabilis lacking flagella cause ascending urinary tract infection in mice.

A clinical isolate of Proteus mirabilis (Pr 990) and an isogenic non-flagellate allelic replacement mutant (Pr M9) were tested for their ability to cause infection in the ascending mouse model of urinary tract infection. Wild-type Pr 990 differentiates into swarmer cells in brain-heart infusion broth. Pr M9 neither has flagella nor does it apparently differentiate into swarmer cells after subculturing. The infectivity of both strains from an initial culture and the sixth subculture was assessed in the ascending urinary tract infection mouse model. Infection was ascertained by determining colony forming units from kidney and bladder homogenates from individual mice 7 days after inoculation. In all cases the nonflagellate mutant Pr M9 was at least as infective as Pr 990. Using bacteria from the first culture, Pr M9 infected 61.5% and Pr 990 infected 45.5% of mice tested. The levels of viable counts were similar between the Pr M9 and the Pr 990 infections. Using bacteria from the sixth subculture, Pr M9 infected 75% and Pr 990 infected 76.5% of mice tested. Again viable counts were similar. Pr 990 increased in infectivity from the first to the sixth subculture, whereas Pr M9 did not, but this may be a reflection of the high initial rate of infectivity with first culture Pr M9. These results suggest that neither flagella nor swarmer cells are required for P. mirabilis infectivity in ascending urinary tract infections in mice.

Alleles↗
PubMed2003

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.

Alleles↗
PubMed2006

Atheroma development in apolipoprotein E-null mice is not regulated by phosphorylation of p27(Kip1) on threonine 187.

Excessive cellular proliferation is thought to contribute to neointimal lesion development during atherosclerosis and restenosis after angioplasty. Inhibition of cyclin-dependent kinase (CDK) activity by p27 inhibits mammalian cell growth. Mounting evidence indicates that p27 negatively regulates neointimal thickening in animal models of restenosis and atherosclerosis, and its expression in human neointimal lesions is consistent with such a protective role. Cell cycle progression is facilitated by cyclinE/CDK2-dependent phosphorylation of p27 on threonine 187 (T187) during late G1. The purpose of this study was to assess whether this phosphorylation event plays a role during atherosclerosis. To this end, we generated apolipoprotein E-null mice with both p27 alleles replaced by a mutated form non-phosphorylatable at T187 (apoE-/-p27T187A mice) and investigated the kinetics of atheroma development in these animals compared to apoE-/- controls with an intact p27 gene. Fat feeding resulted in comparable level of hypercholesterolemia in both groups of mice. Surprisingly, aortic p27 expression was not increased in fat-fed apoE-/-p27T187A mice compared with apoE-/- controls. Moreover, atheroma size, lesion cellularity, proliferation, and apoptotic rates were undistinguishable in both groups of fat-fed mice. Thus, in contrast to previous studies that highlight the importance of p27 phosphorylation at T187 on the control of p27 expression and function in different tissues and pathophysiological scenarios, our findings demonstrate that this phosphorylation event is not implicated in the control of aortic p27 expression and atheroma progression in hypercholesterolemic mice.

Animals↗
PubMed2000

Expression and regulation of the streptokinase gene.

Recent research in various areas has appreciably expanded our knowledge of streptokinase, a plasminogen activator produced by all human group A (GAS), group C (GCS), and group G (GGS) streptococci. Several molecular genetic approaches are described here to study the expression of the streptokinase gene, skn. Southern hybridization analysis demonstrated homology of synteny of ska, skc, and skg in the genomes of the above serogroups. S1 nuclease mapping, the use of transcriptional fusions to beta-galactosidase and luciferase reporter genes, in conjunction with site-directed mutagenesis, led to the localization of the core promoter region of skc and the identification of a cis-active upstream region required for full promoter activity. Circular permutation analysis of the promoter upstream region identified an intrinsic DNA bending locus as the pivotal DNA element stimulating the activity of the core promoter. The detection of skn allele-specific expression phenotypes, which proved not to be due to different skn mRNA half-lives, prompted allele swap experiments, showing that promoter activity is dictated by the host genetic background, rather than the sequence of the regulatory region. These findings suggest the involvement in skn expression of an as yet unidentified transcriptional activator that contacts the bent DNA region. Transcription termination of skc is directed by a bidirectional terminator whose structural requirements for termination efficiency were determined with base substitution mutants fused to a chloramphenicol acetyl transferase reporter. Finally, mutagenic plasmids are described for insertion-duplication and allele replacement mutagenesis of the skn locus.

Base Sequence↗
PubMed1998

Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence.

Allelic replacement was used to generate two isogenic lktA deletion mutants of Pasteurella haemolytica serotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed that lktA sequences were absent in the two P. haemolytica deletion mutants. Culture supernatants and whole cell lysates from the wild type P. haemolytica, D153 parent strain, but not the lktA deletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of the lktA deletion mutant, lktA 77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in the lktA mutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with the lktA mutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Animals↗
PubMed1998

Assessment of the role of gamma-toxin in experimental endophthalmitis using a hlg-deficient mutant of Staphylococcus aureus.

Purified gamma-toxin is known to have a proinflammatory effect in the rabbit vitreous humor. To assess the biological role of the gamma-toxin, when expressed in vivo by Staphylococcus aureus strain Newman, the vitreous humor of rabbit eye was used as an infection model. A gamma-toxin-deficient mutant of strain Newman was constructed by allelic replacement. S. aureus Newman wild-type, its hlg-deficient derivative strain (N65) and the strain N65 complemented with the wild-type hlg+ gene were injected into the vitreous humor of rabbit eye. All three strains produced a strong proinflammatory effect in the eye conjunctiva, posterior and anterior chambers, suggesting a role for another unidentified proinflammatory component of strain Newman distinct from the gamma-toxin. These components are not the leucocidin of Panton-Valentine, beta-toxin or alpha-toxin which are not produced by this strain. Only the hlg-deficient mutant lacked the ability to cause inflammation in the eyelid, whereas the two Hlg-producing strains gave strong inflammation. These data suggest that in vivo, strain Newman produces as yet unidentified proinflammatory molecules and that the in vivo-produced HlgA, HlgB and HlgC molecules expressed by the gamma-toxin locus, contribute in part to the inflammatory process observed in vivo in the rabbit eye.

Animals↗
PubMed1999

Capsular hyaluronic acid of group A streptococci hampers their invasion into human pharyngeal epithelial cells.

Group A streptococci (GAS) cause various diseases, from uncomplicated noninvasive, to severe invasive infections. Capsular hyaluronic acid (HA) is known to resist phagocytosis, however, interaction between HA and epithelial cells have not been clearly understood. In this study, both HA-producing wild strains and HA-nonproducing mutants were employed to examine their invasiveness into confluent cultures of HEp-2, a nonphagocytic human epithelial cell line. Invasion of HEp-2 cells by GAS strains increased over time. The hasA gene encoding hyaluronate synthase of GAS strains was inactivated by allelic replacement. It was found that hasA-inactivated mutants were internalized into HEp-2 cells more efficiently than their parent strains under various conditions in terms of incubation time and inoculum size. Taken together, these findings indicate that GAS can be internalized into HEp-2 cells with considerably high frequencies and that the presence of HA of GAS decreased the invasion efficiency.

Animals↗
PubMed2000

Transcription of the transfer genes traY and traM of the antibiotic resistance plasmid R100-1 is linked.

Three separate traY deletion mutants of R100-1 were prepared by allele replacement. These mutants retained the ability to transfer at a level 100 times greater than R100 and 1/50 that of the parental R100-1. The mutants were complemented to normal R100-1 transfer levels by pDSP06, a multicopy traY clone. Comparison of transcripts initiated at the traY promoter, P(Y), by primer extension experiments showed that there was no detectable P(Y) activity in R100 and that the level of P(Y) activity in the traY deletion mutants was lower than that in R100-1. Similar measurements performed on RNA from a set of previously described traM deletion mutants showed that those traM deletion mutants that produced more traM and finM (M) transcripts than the parental R100-1 also produced more traY transcripts than R100-1 and that those traM mutants that produced fewer M transcripts than R100-1 also produced fewer traY transcripts than R100-1. We conclude that in R100, TraY regulates P(Y) activity and that transcripts originating in traM affect P(Y) activity.

Amino Acid Sequence↗
PubMed1989

Vitamin B12 transport in Escherichia coli K12 does not require the btuE gene of the btuCED operon.

Transport of vitamin B12 across the cytoplasmic membrane of Escherichia coli requires the products of btuC and btuD, two genes in the btuCED operon. The role of btuE, the central gene of this operon, was examined. Deletions within btuE were constructed by removal of internal restriction fragments and were crossed onto the chromosome by allelic replacement. In-frame deletions that removed 20% or 82% of the btuE coding region did not affect expression of the distal btuD gene. These nonpolar deletions had little effect on vitamin B12 binding (whole cells or periplasmic fraction) and transport. They did not affect the utilization of vitamin B12 or other cobalamins for methionine biosynthesis, even in strains with decreased outer membrane transport of vitamin B12. The btuE mutations did not impair adenosyl-cobalamin dependent catabolism of ethanolamine or repression of btuB expression. Thus, despite its genetic location in the transport operon, the btuE product plays no essential role in vitamin B12 transport.

Amino Acid Sequence↗
PubMed2002

A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant.

Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.

Aspartate Kinase↗
PubMed2006

A leuC mutation leading to increased L-lysine production and rel-independent global expression changes in Corynebacterium glutamicum.

We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner.

Corynebacterium glutamicum↗
PubMed1992

Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis.

The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.

Alkaline Phosphatase↗
PubMed1983

Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon.

A restriction map was determined for a phi 80 lambda dhis transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon (hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI-EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp::his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp::his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.

Bacteriophages↗
PubMed1988

Expression of human papillomavirus type 6b E2 gene product with DNA-binding activity in insect (Bombyx mori) cells using a baculovirus expression vector.

Human papillomavirus type 6b (HPV6b) has been shown to be a major etiologic agent of genital condylomas. The E2 gene, one of the early genes, has been shown to activate the enhancer element in trans in cells transformed with bovine papillomavirus type 1a (BPV1a) but the E2 gene product of any HPV has not been identified. The E2 gene of HPV6b was inserted into the polyhedrin gene of a Baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), 156 bp downstream from the translational start codon, and transferred into BmNPV by allelic replacement in a cotransfected Bombyx mori cell line, Bm-N. The predicted 46-kDa protein was produced by a recombinant virus in the infected Bm-N cells at a high level under the control of the polyhedrin promoter. We obtained the antibody against the putative E2-polyhedrin fusion protein by immunizing a rabbit with this protein. This protein reacted with the antibodies against polyhedrin and the fusion protein. This protein did specifically bind to the upstream regulatory region of the HPV6b and BPV1a genomes. This DNA binding activity was blocked by the antibody against this protein.

Animals↗