Home About us Contact
|
Home About us Contact | ||
|
|
||
Transposon Mutant (transposon + mutant)
Selected AbstractsRole of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12ENVIRONMENTAL MICROBIOLOGY, Issue 1 2009Jincai Ma Summary In this study, we investigated the role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. A mini-Tn5 transposon mutant of E. cloacae SLD1a-1, designated as 4E6, was isolated that had lost the ability to reduce Se(VI) to Se(0). Genetic analysis of mutant strain 4E6 showed that the transposon was inserted within a menD gene among a menFDHBCE gene cluster that encodes for proteins required for menaquinone biosynthesis. A group of E. coli K12 strains with single mutations in the menF, menD, menC and menE genes were tested for loss of selenate reduction activity. The results showed that E. coli K12 carrying a deletion of either the menD, menC or menE gene was unable to reduce selenate. Complementation using wild-type sequences of the E. cloacae SLD1a-1 menFDHBCE sequence successfully restored the selenate reduction activity in mutant strain 4E6, and E. coli K12 menD and menE mutants. Selenate reduction activity in 4E6 was also restored by chemical complementation using the menaquinone precursor compound 1,4-dihydroxy-2-nathphoic acid. The results of this work suggest that menaquinones are an important source of electrons for the selenate reductase, and are required for selenate reduction activity in E. cloacae SLD1a-1 and E. coli K12. [source] Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunityEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Christy Abstract Francisella tularensis (FT) has been classified by the CDC as a category,A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAgneg) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies. [source] Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting uptake of dibenzothiophene in n -tetradecaneLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2003K. Noda Abstract Aims: Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting the uptake of dibenzothiophene (DBT) in n -tetradecane (n -TD). Methods and Results: The dsz desulphurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred to the chromosome of P. aeruginosa NCIMB9571 using a transposon vector. A recombinant (named PARM1) was obtained which was able to desulphurize DBT in water, but not in n -TD. Conclusions: PARM1 is a mutant deficient in a DBT transport system operational in n -TD. This transport system is independent of rhamnolipids and of the n -alkane transport system. Significance and Impact of the Study:Pseudomonas aeruginosa NCIMB9571 seems to have a specific system of transporting hydrophobic compounds such as DBT in oil. [source] Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilmsMOLECULAR MICROBIOLOGY, Issue 1 2004Irene Kuiper Summary Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High-performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface-tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface-active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N-terminus of a 12-amino-acid peptide moiety, in which the C-terminal carboxylic acid group forms an ester with the hydroxyl side-chain of Ser9. The difference between the two structures is located in the second amino acid from the C-terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio-synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild-type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms. [source] Characterization of RAT, an autolysis regulator in Staphylococcus aureusMOLECULAR MICROBIOLOGY, Issue 6 2003S. S. Ingavale Summary In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus. [source] Overexpression of hns in the plant growth-promoting bacterium Enterobacter cloacae UW5 increases root colonizationJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2010M.M. English Abstract Aims:, Plant growth-promoting rhizobacteria (PGPR) introduced into soil often do not compete effectively with indigenous micro-organisms for plant colonization. The aim of this study was to identify novel genes that are important for root colonization by the PGPR Enterobacter cloacae UW5. Methods and Results:, A library of transposon mutants of Ent. cloacae UW5 was screened for mutants with altered ability to colonize canola roots using a thermal asymmetric interlaced (TAIL)-PCR-based approach. A PCR fragment from one mutant was reproducibly amplified at greater levels from genomic DNA extracted from mutant pools recovered from seedling roots 6 days after seed inoculation compared to that from the cognate inoculum cultures. Competition assays confirmed that the purified mutant designated Ent. cloacae J28 outcompetes the wild-type strain on roots but not in liquid cultures. In Ent. cloacae J28, the transposon is inserted upstream of the hns gene. Quantitative RT-PCR showed that transposon insertion increased expression of hns on roots. Conclusions:, These results indicate that increased expression of hns in Ent. cloacae enhances competitive colonization of roots. Significance and Impact of the Study:, A better understanding of the genes involved in plant colonization will contribute to the development of PGPR that can compete more effectively in agricultural soils. [source] A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretionMOLECULAR MICROBIOLOGY, Issue 6 2004Lian-Yong Gao Summary Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871,Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host. [source] Identification of Xenorhabdus nematophila genes required for mutualistic colonization of Steinernema carpocapsae nematodesMOLECULAR MICROBIOLOGY, Issue 5 2002Kurt Heungens Summary One stage in the symbiotic interaction between the bacterium Xenorhabdus nematophila and its nematode host, Steinernema carpocapsae, involves the species-specific colonization of the nematode intestinal vesicle by the bacterium. To characterize the bacterial molecular determinants that are essential for vesicle colonization, we adapted and applied a signature-tagged mutagenesis (STM) screen to this system. We identified 15 out of 3000 transposon mutants of X. nematophila with at least a 15-fold reduction in average vesicle colonization. These 15 mutants harbour disruptions in nine separate loci. Three of these loci have predicted open reading frames (ORFs) with similarity to genes (rpoS, rpoE, lrp) encoding regulatory proteins; two have predicted ORFs with similarity to genes (aroA, serC) encoding amino acid biosynthetic enzymes; one, designated nilB (nematode intestine localization), has an ORF with similarity to a gene encoding a putative outer membrane protein (OmpU) in Neisseria; and three, nilA, nilC and nilD, have no apparent homologues in the public database. nilA, nilB and nilC are linked on a single 4 kb locus. nilB and nilC are > 104 -fold reduced in their ability to colonize the nematode vesicle and are predicted to encode membrane-localized proteins. The nilD locus contains an extensive repeat region and several small putative ORFs. Other than reduced colonization, the nilB, nilC and nilD mutants did not display alterations in any other phenotype tested, suggesting a specific role for these genes in allowing X. nematophila to associate with the nematode host. [source] Multiple determinants influence root colonization and induction of induced systemic resistance by Pseudomonas chlororaphis O6MOLECULAR PLANT PATHOLOGY, Issue 6 2006SONG HEE HAN SUMMARY Colonization of the roots of tobacco by Pseudomonas chlororaphis O6 induces systemic resistance to the soft-rot pathogen, Erwinia carotovora ssp. carotovara SCC1. A screen of the transposon mutants of P. chlororaphis O6 showed mutants with about a fivefold reduction in ability to induce systemic resistance to the soft-rot disease. These mutations disrupted genes involved in diverse functions: a methyl-accepting chemotaxis protein, biosynthesis of purines, phospholipase C, transport of branched-chain amino acids and an ABC transporter. Additional mutations were detected in the intergenic spacer regions between genes encoding a GGDEF protein and fumarate dehydratase, and in genes of unknown function. The mutants in the ABC transporters did not display reduced root colonization. However, the other mutants had up to 100-fold reduced colonization levels. Generally the production of metabolites important for interactions in the rhizosphere, phenazines and siderophores, was not altered by the mutations. A reduced induction of systemic resistance by a purine biosynthesis mutant with a disrupted purM gene correlated with poor growth rate, lesser production of phenazines and siderophore and low levels of root colonization. These studies showed that multiple determinants are involved in the induction of systemic resistance, with there being a requirement for strong root colonization. [source] Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbationsBIOTECHNOLOGY PROGRESS, Issue 5 2009Yinjie J. Tang Abstract An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time ,3 h), salt stressed minimal medium (doubling time ,6 h), and minimal medium with amino acid supplementation (doubling time ,1.5 h). 13C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, 10 transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||