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Mutant Strain (mutant + strain)
Terms modified by Mutant Strain Selected AbstractsSynthesis of Novel Diarylpyrimidine Analogues of TMC278 and Their Antiviral Activity Against HIV-1 Wild-Type and Mutant Strains.CHEMINFORM, Issue 40 2007Jerome Guillemont Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Escherichia coli,-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocationCELLULAR MICROBIOLOGY, Issue 10 2007Hanno Troeger Summary Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (Rt) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an Rt decrease (36 ± 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-, or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased Rt and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli,-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines. [source] Survival of Erwinia amylovora on pears and on fruit containers in cold storage and outdoorsEPPO BULLETIN, Issue 1 2004P. Ceroni The survival of Erwinia amylovora during cold storage or outdoors may be a relevant factor in the spread of fireblight. The survival of E. amylovora was studied in cold storage on pear fruits, on container materials and on packaging paper, and outdoors on wood (oak and poplar) and on polyethylene. The samples were contaminated with a bacterial suspension of a mutant strain, washed, concentrated by centrifugation, and the final concentrates were used for plate counting. In cold storage, reisolation from the calyx was successful even after 101 days, whereas on pear surfaces, it was unsuccessful after just 1 day. On oak and poplar wood, reisolation was obtained up to 77 days in cold storage for both types of wood, but only up to 27 and 55 days, respectively, outdoors. Reisolation from packaging paper in cold storage was successful up to 14 days. Reisolation from polyethylene outdoors was unsuccessful after 24 h. Survival curves were calculated for each material. On the basis of a model of inoculum transmission, and using the survival curves, a phytosanitary risk period for the different types of materials was estimated. [source] Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteriaFEBS JOURNAL, Issue 15 2007María R. Gómez-García Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1) were isolated and characterized from three phylogenetically diverse cyanobacteria , Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Pseudanabaena sp. PCC 6903 , and one anoxygenic photosynthetic bacterium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were found to be family I soluble inorganic pyrophosphatases with c. 20 kDa subunits with diverse oligomeric structures. The corresponding ppa genes were cloned and functionally validated by heterologous expression. Cyanobacterial family I soluble inorganic pyrophosphatases were strictly Mg2+ -dependent enzymes. However, diverse cation cofactor dependence was observed for enzymes from other groups of photosynthetic bacteria. Immunochemical studies with antibodies to cyanobacterial soluble inorganic pyrophosphatases showed crossreaction with orthologs of other main groups of phototrophic prokaryotes and suggested a close relationship with the enzyme of heliobacteria, the nearest photosynthetic relatives of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain, containing a very low level of soluble inorganic pyrophosphatase activity, was functionally complemented up to wild-type growth rates with ppa genes from diverse photosynthetic prokaryotes expressed under their own promoters. Overall, these results suggest that the bacterial family I soluble inorganic pyrophosphatases described here have retained functional similarities despite their genealogies and their adaptations to diverse metabolic scenarios. [source] 7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic targetFEBS JOURNAL, Issue 20 2006Characterization, inhibition studies Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S -adenosyl- l -methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5,-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pKa of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: KmAdoMet = 0.78 ± 0.20 mm, KmKAPA = 3.8 ± 1.0 µm, kcat = 1.0 ± 0.2 min,1, KiKAPA = 14 ± 2 µm. Amiclenomycin and a new analogue, 4-(4c -aminocyclohexa-2,5-dien-1r -yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: Ki = 12 ± 2 µm, kinact = 0.35 ± 0.05 min,1, and Ki = 20 ± 2 µm, kinact = 0.56 ± 0.05 min,1, for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 µg·mL,1) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo. [source] Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNAFEBS JOURNAL, Issue 20 2005Ernesto I. Gonzalez De Valdivia In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expression of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibitory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger peptidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later. [source] Defective translocation of a signal sequence mutant in a prlA4 suppressor strain of Escherichia coliFEBS JOURNAL, Issue 22 2002Hendrik Adams In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem.269, 5564,5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position ,10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon. [source] Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp. citri: targeting the division septumFEMS MICROBIOLOGY LETTERS, Issue 1 2010Paula M.M. Martins Abstract Xanthomonas citri ssp. citri (Xac) is the causal agent of citrus canker, an economically important disease that affects citrus worldwide. To initiate the characterization of essential biological processes of Xac, we constructed integrative plasmids for the ectopic expression of green fluorescent protein (GFP)-labeled proteins within this bacterium. Here, we show that the disruption of the ,-amylase gene (amy), the site of plasmid integration into the bacterial chromosome, does not alter its pathogenesis while abolishing completely the ability of Xac to degrade starch. Furthermore, our GFP expression system was used to characterize ORF XAC3408, a hypothetical protein encoded by Xac that shares significant homology to the FtsZ-stabilizing factor ZapA from Bacillus subtilis (ZapABsu). GFP-XAC3408 expressed in Xac exhibited a septal localization pattern typical of GFP-ZapABsu, which indicates that XAC3408 is the Xac orthologue of the cell division protein ZapABsu. The results demonstrate the potential of GFP labeling for protein functional characterizations in Xac, and, in addition, the Xac mutant strain labeled at the septum constitutes a biological model for the exploration of antibacterial compounds able to inhibit cell division in this plant pathogen. [source] Cloning and characterization of CmGPD1, the Candida magnoliae homologue of glycerol-3-phosphate dehydrogenaseFEMS YEAST RESEARCH, Issue 8 2008Dae-Hee Lee Abstract Glycerol-3-phosphate dehydrogenase (GPDH) plays a central role in glycerol metabolism. A genomic CmGPD1 gene encoding NADH-dependent GPDH was isolated from Candida magnoliae producing a significant amount of glycerol. The gene encodes a polypeptide of 360 amino acids, which shows high homology with known NADH-dependent GPDHs of other species. The CmGPD1 gene was expressed in recombinant Escherichia coli with the maltose-binding protein (MBP) fusion system and purified to homogeneity using simple affinity chromatography. The purified CmGpd1p without the MBP fusion displayed an apparent molecular mass of 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The CmGpd1p enzyme exhibited a Kcat/Km value of 195 min,1 mM,1 for dihydroxyacetone phosphate whereas Kcat/Km for glycerol-3-phosphate is 0.385 min,1 mM,1. In a complementation study, CmGpd1p rescued the ability of glycerol synthesis and salt tolerance in a Saccharomyces cerevisiae GPD1,GPD2, mutant strain. The overall results indicated that CmGPD1 encodes a functional homologue of S. cerevisiae GPDH. [source] Arsenic induces caspase- and mitochondria-mediated apoptosis in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 6 2007Li Du Abstract In recent years, it has been shown that yeast, a unicellular organism, undergoes apoptosis in response to various factors. Here we demonstrate that the highly effective anticancer agent arsenic induces apoptotic process in yeast cells. Reactive oxygen species (ROS) production was observed in the process. Moreover, mitochondrial membrane potential decreased after arsenic treatment. Resistance of the rho0 mutant strain (lacking mtDNA) to arsenic provides further evidence that this death process involves mitochondria. In addition, hypersensitivity of ,sod1 to arsenic suggests the critical role of ROS. Cell death and DNA fragmentation decreased in a ,yca1 deletion mutant, indicating the participation of yeast caspase-1 protein in apoptosis. The implications of these findings for arsenic-induced apoptosis are discussed. [source] Involvement of RNase G in in vivo mRNA metabolism in Escherichia coliGENES TO CELLS, Issue 5 2001Genryou Umitsuki Background Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5, end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. Results We found that rng::cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng::cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng::cat disruptant than in the wild-type. Conclusion These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli. [source] Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti-terminator BglG, CRP-cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiationGENES TO CELLS, Issue 4 2000Abhilasha Gulati Background Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic ,-glucosides, is extremely sensitive to the presence of glucose in the growth medium. We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression. Results Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose. Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho-independent terminator present within the leader sequence. Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose-mediated repression is the anti-terminator protein, BglG. Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression. Studies using crp, cya and crr strains show that both CRP-cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation. Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator. Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose-grown cultures of a crp+ strain. Glucose repression is lost in the crr mutant strain. Conclusions The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti-termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF. The CRP-cAMP complex is also involved in this regulation. The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes. [source] Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68GENES TO CELLS, Issue 4 2000Seung-Woo Kang Background Transcriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE-interacting factors which have chromatin-related functions. Results Using the yeast two-hybrid screening system, we isolated the C-terminal part of the human homologue of Saccharomyces cerevisiae (y) Spt16p/Cdc68p, a general chromatin factor. The C-terminal part of human SPT16/CDC68 directly interacts with TFIIE, and ySpt16p/Cdc68p also interacts with yTFIIE (Tfa1p/Tfa2p), thus indicating the existence of an evolutionarily conserved interaction between TFIIE and SPT16/CDC68. Functional interaction of yTFIIE and ySpt16p/Cdc68p was examined using a conditional yTFIIE-, mutant strain. Over-expression of ySpt16p/Cdc68p suppressed the phenotype of cold sensitivity of the yTFIIE-,- cs mutant strain, and in vitro binding assays revealed that yTFIIE-,- cs mutant protein showed diminished binding affinity to ySpt16p/Cdc68p. Conclusions These observations indicate that general transcription initiation factor TFIIE functionally interacts with general chromatin factor SPT16/CDC68, a finding which provides new insight into the involvement of TFIIE in chromatin transcription. This may well lead to a breakthrough in relationships between the transcription initiation process and structural changes in chromatin. [source] Towards a molecular definition of worker sterility: differential gene expression and reproductive plasticity in honey beesINSECT MOLECULAR BIOLOGY, Issue 5 2006G. J. Thompson Abstract We show that differences in the reproductive development of honey bee workers are associated with locus-specific changes to abundance of messenger RNA. Using a cross-fostering field experiment to control for differences related to age and environment, we compared the gene expression profiles of functionally sterile workers (wild-type) and those from a mutant strain in which workers are reproductively active (anarchist). Among the set of three genes that are significantly differentially expressed are two major royal jelly proteins that are up-regulated in wild-type heads. This discovery is consistent with sterile workers synthesizing royal jelly as food for developing brood. Likewise, the relative underexpression of these two royal jellies in anarchist workers is consistent with these workers' characteristic avoidance of alloparental behaviour, in favour of selfish egg-laying. Overall, there is a trend for the most differentially expressed genes to be up-regulated in wild-type workers. This pattern suggests that functional sterility in honey bee workers may generally involve the expression of a suite of genes that effectively ,switch' ovaries off, and that selfish reproduction in honey bee workers, though rare, is the default developmental pathway that results when ovary activation is not suppressed. [source] The role of GAP1 gene in the nitrogen metabolism of Saccharomyces cerevisiae during wine fermentationJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009R. Chiva Abstract Aim:, The aim of this study was to analyse the relevance of the general amino acid permease gene (GAP1) of the wine yeast Saccharomyces cerevisiae on nitrogen metabolism and fermentation performance. Methods and Results:, We constructed a gap1 mutant in a wine strain. We compared fermentation rate, biomass production and nitrogen consumption between the gap1 mutant and its parental strain during fermentations with different nitrogen concentrations. The fermentation capacity of the gap1 mutant strain was impaired in the nitrogen-limited and -excessive conditions. The nitrogen consumption rate between the wild strain and the mutant was different for some amino acids, especially those affected by nitrogen catabolite repression (NCR). The deletion of GAP1 gene also modified the gene expression of other permeases. Conclusions:, The Gap1 permease seems to be important during wine fermentations with low and high nitrogen content, not only because of its amino acid transporter role but also because of its function as an amino acid sensor. Significance and Impact of the Study:, A possible biotechnological advantage of a gap1 mutant is its scarce consumption of arginine, whose metabolism has been related to the production of the carcinogenic ethyl carbamate. [source] Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2001F. Mozzi Aims: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined. Methods and Results: The concentration of EPS was estimated by the phenol/sulphuric acid method while the chemical composition of purified EPS was investigated using gas-liquid chromatography. Biosynthetic enzyme activities were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. Polysaccharide production by Lb. casei CRL 87 was 1·7 times greater on galactose than on glucose. The isolated polymer was composed of rhamnose, glucose and galactose. The activities of uridine-diphosphate (UDP)-glucose-pyrophosphorylase, thymidine-diphosphate (dTDP)-glucose-pyrophosphorylase and the dTDP-rhamnose-synthetic enzyme system were higher in galactose-grown than in glucose-grown cells. When an EPS, mutant strain was used, galactokinase activity was not detected on galactose, this sugar not being available for the formation of sugar nucleotides for further EPS production. dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities were lower than the values found for the wild type strain. Conclusions: The carbon source present in the culture medium affects EPS production by Lb. casei CRL 87. The greater polymer synthesis by galactose-grown cells is correlated with the higher UDP-glucose-pyrophosphorylase, dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities. Initial sugar metabolism is also an important step for the synthesis of EPS precursors by this strain. Significance and Impact of the Study: Knowledge of the effect of the sugar source on EPS production and the activities of biosynthetic enzymes provides information about the mechanisms of regulation of the synthesis of EPS which can contribute to improving polymer production. [source] Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistanceJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000J.P. Grill Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N,-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (Z,pH) was involved in Lactobacillus bile salt resistance. [source] Transcription factor Stb5p is essential for acetaldehyde tolerance in Saccharomyces cerevisiaeJOURNAL OF BASIC MICROBIOLOGY, Issue 5 2010Yoshimi Matsufuji Abstract Transcription factor Stb5p, previously known as one of the multidrug resistance gene regulators in Saccharomyces cerevisiae, was shown here to play an essential role in acetaldehyde tolerance. A mutant strain, ,stb5 exhibited increased acetaldehyde sensitivity, and failed to induce genes such as GND1, TKL1 and TAL1 involved in the pentose phosphate pathway (PPP) upon acetaldehyde stress. Using this strain it was revealed that Stb5p acts as a repressor for PGI1 encoding glucose-6-phosphate isomerase under acetaldehyde stress. In reverse, over-expression of Stb5p reinforced acetaldehyde tolerance to the yeast. Furthermore, various deletion mutants of the genes involved in glycolysis showed increased acetaldehyde tolerance compared to the wild-type strain. From these results, it was suggested that Stb5p participates in acetaldehyde tolerance by regulating expression of the PPP genes and PGI1, and that down-regulation of glycolytic pathway may lead to vitalization of PPP and to increased acetaldehyde tolerance. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulansJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 4 2006S. E. SCHOUSTRA Abstract We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans. [source] THE INFLUENCE OF SAE LOCUS KNOCKOUT ON EXOPROTEINS IN STAPHYLOCOCCUS AUREUSJOURNAL OF FOOD SAFETY, Issue 3 2010JUNNI TANG ABSTRACT The sae operon is a key regulator in Staphylococcus aureus, which is known as an important infective and toxigenic bacterial pathogen. For the exploration of virulence factors expressed in the secreted exoprotein fraction are being controlled by sae operon, the relationship between the sae locus and exoproteins was investigated in this study. The homologous recombination vector pBT2,sae was constructed and the sae deletion mutant strain was successfully obtained. The results showed that the sae locus played an important role in the production of thermonucleases and other exoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed different exoprotein profiles between parent strain and mutant strain, in which three bands were visibly weakened. The results revealed that sae locus was involved in the regulation on exoproteins, some of which play a known fundamental role in the virulence of S. aureus. PRACTICAL APPLICATIONS This study presents that knocking out the sae gene locus in a specific Staphylococcus aureus strain results in reduced thermonuclease action, and also in reduced levels of proteins in the vicinity of 42 and 32 kDa molecular weight in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels, indicating that their production is dependent on the sae locus. Practically, these proteins are associated with virulence traits, and with the pathogen's response to the environment and in potential hosts, which could be helpful for understanding the pathogenicity of S. aureus and also for further studies on the role of selected genes in the pathogenicity of S. aureus. [source] Optimization of Medium Composition for Nisin Fermentation with Response Surface MethodologyJOURNAL OF FOOD SCIENCE, Issue 6 2008X.-X. Zhou ABSTRACT:, Nisin is an effective food biopreservative widely used in food industry. However, 1 problem of concern is limited production rate and final nisin concentration. A nisin-producing strain, L. lactis Lac2, a mutant strain with high yield of nisin, was obtained in our laboratory recently. In the present study, a fractional factorial design was applied to investigate the main factors that affect the yield of L. lactis Lac2. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The results showed that the optimum medium for nisin production of L. lactis Lac2 was composed of 2.68% sucrose (w/v), 0.5% tryptone (w/v), 1% yeast extract (w/v), 0.3% Tween-80 (w/v), 0.02% MgSO4·7H2O (w/v), 0.81% NaCl (w/v), 1.91% K2HPO4 (w/v), 0.05% ascorbic acid (w/v), and 2% agar (w/v) (if necessary) at pH 6.5. When cultured in the optimum medium, the nisin yield is an average of 3381.81 IU/mL, which nearly doubled the yield when incubated in the initial medium. Also, the concentration of tryptone was decreased while that of the sucrose was increased when compared with CM broth, which means a reduction of the fermentation cost. [source] Successful treatment of an entecavir-resistant hepatitis B virus variantJOURNAL OF MEDICAL VIROLOGY, Issue 12 2007Hiromi Yatsuji Abstract Emergence of a lamivudine (LAM)-resistant hepatitis B virus (HBV) with amino acid substitutions in the YMDD motif is a well-documented problem during long-term LAM therapy. Entecavir (ETV) is a new drug approved for treatment of HBV infection with or without LAM-resistant mutants. This report describes an ETV-resistant strain of HBV, which emerged after prolonged ETV therapy in a patient who did not respond to LAM therapy. Direct sequence analysis of the ETV-resistant strain showed appearance of amino acid substitution rtS202G in the reverse transcriptase (RT) domain, together with rtL180M,+,M204V substitution that had developed at the emergence of LAM-resistant mutant. In vitro analysis demonstrated that the rtL180M,+,M204V,+,S202G mutant strain displayed a 200-fold and a 5-fold reduction in susceptibility to ETV compared with the wild- type and the rtL180M,+,M204V mutant strain, respectively. Adefovir was effective against the ETV-resistant strain both in vitro and during the clinical course. In conclusion, this study showed that virological and biochemical breakthrough due to ETV could occur in patients infected with LAM-resistant HBV and confirmed that the addition of rtS202G substitution to the rtL180M,+,M204V mutant strain is responsible for ETV resistance and we could treat the resistant mutant successfully. J. Med. Virol. 79:1811,1817, 2007. © 2007 Wiley-Liss, Inc. [source] Dense fimbrial meshwork enhances Porphyromonas gingivalis adhesiveness: a scanning electron microscopic studyJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2007H. Hongo Background and Objective:, The aim of this study was to determine how the fimbriae of Porphyromonas gingivalis function in plaque formation. Material and Methods:, We used scanning electron microscopy to examine aggregates and hemaggregates of fimbria-rich ATCC33277 (parent) and fimbra-poor OZ6301C (pgmA -knockout, mutant) strains of P. gingivalis. We also assessed the hemagglutination activity of the two strains as an indicator of P. gingivalis adhesiveness. Results:, Aggregates of P. gingivalis were composed of bacterial chains and clusters. Rich fimbriae projecting from cells of the parent strain tended to bunch and form a dense meshwork among bacterial cells. In contrast, cells of the mutant strain projected fewer fimbriae and the meshwork was looser. Hemaggregates including cells of the parent strain contained a detached, dense fimbrial meshwork that adhered to erythrocytes. Hemaggregates comprising cells of the mutant strain included bacterial chains and clusters that adhered to erythrocytes by shorter fimbriae than those of the parent strain. The hemagglutination titer of the parent strain was 10-fold higher than that of the mutant strain, although the number of fimbriae per cell of the parent strain was only double that of the mutant strain. Conclusion:, The results indicate that P. gingivalis adhesiveness is prominently enhanced by the dense fimbrial meshwork. Thus, the virulence of P. gingivalis is increased by the presence of rich fimbriae. [source] ISOLATION AND CHARACTERIZATION OF A CELL WALL-DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA),JOURNAL OF PHYCOLOGY, Issue 6 2003Cesar Fuentes Cell wall,defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw-1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non-ionic detergent as compared with wild-type cells. Tetrad analysis of crosses involving the cw-1 mutant confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw-1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw-1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols. [source] Lack of O -polysaccharide enhances biofilm formation by Bradyrhizobium japonicumLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2010Y.-W. Lee Abstract Aims:, To reveal the effects of the O -polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. Methods and Results:, Wild type and O-antigen-deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi-solid (0·3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3·5-fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. Conclusions:, This study shows enhanced biofilm formation and decreased motility by the O-antigen-deficient mutant, suggesting that the lack of the O -polysaccharide of the rhizobial LPS is associated with biofilm-forming ability and movement. Significance and Impact of the Study:, LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O-antigen-deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum. [source] Regulation of pyrimidine nucleotide formation in Pseudomonas reptilivoraLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2004T.P. West Abstract Aims:, To study the regulation of de novo pyrimidine biosynthesis in the pathogenic bacterium Pseudomonas reptilivora ATCC 14836. Methods and Results:, The pyrimidine biosynthetic pathway enzymes were assayed in extracts of Ps. reptilivora ATCC 14836 cells and of cells from an auxotroph lacking aspartate transcarbamoylase activity. Pyrimidine biosynthetic pathway enzyme activities in ATCC 14836 were influenced by the addition of pyrimidine bases to the culture medium with orotic acid addition inducing dihydroorotase activity. Pyrimidine starvation of the transcarbamoylase mutant strain increased its de novo enzyme activities suggesting that the de novo pathway was also subject to repression by a pyrimidine-related compound. Aspartate transcarbamoylase activity in ATCC 14836 was inhibited in vitro by pyrophosphate and ATP. Conclusions:, Regulation of pyrimidine biosynthesis in Ps. reptilivora was observed at the level of enzyme synthesis and at the level of activity for aspartate transcarbamoylase. Its regulation of enzyme synthesis seemed to be more highly controlled than what was observed in the related species Ps. fluorescens. Significance and Impact of the Study:, This investigation found that pyrimidine biosynthesis is controlled in Ps. reptilivora. This could prove helpful to future studies exploring its pathogenicity. [source] Functional specialization and differential regulation of short-chain carboxylic acid transporters in the pathogen Candida albicansMOLECULAR MICROBIOLOGY, Issue 6 2010Neide Vieira Summary The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1-GFP (green fluorescent protein) and Jen2-GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1-GFP fusion. In the murine model of systemic candidiasis approximately 20,25% of C. albicans cells infecting the kidney expressed Jen1-GFP and Jen2-GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose-poor niches within the host, and that these short-chain carboxylic acid transporters may be important in the early stages of infection. [source] Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriaeMOLECULAR MICROBIOLOGY, Issue 4 2007Anu Swaminathan Summary Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres , SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase. [source] Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowthMOLECULAR MICROBIOLOGY, Issue 2 2007Christopher T. Steichen Summary Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is formed by a collagen-like glycoprotein called BclA, while the basal layer contains many different proteins, one of which is a spore-specific alanine racemase (Alr). In this study, we employed fluorescence microscopy and a fluorescently labelled anti-Alr monoclonal antibody (mAb) to examine the distribution of Alr within the exosporium. Binding of the mAb occurred over approximately three-quarters of the exosporium but not in a cap-like region at one end of the spore, indicating the absence or inaccessibility of Alr in this region. We also determined that the cap-like region, or cap, corresponds to the first part of the exosporium assembled within the mother cell during sporulation and the only part of the exosporium assembled in a ,exsY mutant strain of B. anthracis. Our results provide the first direct evidence that exosporium assembly is a non-uniform process and suggest that exosporium formation is discontinuous. Finally, we demonstrated that during spore germination and outgrowth, the outgrowing cell always escapes from its exosporium shell by popping through the cap, suggesting that the cap is designed to facilitate the emergence of the outgrowing cell. [source] Positive regulation of Bacillus subtilis ackA by CodY and CcpA: establishing a potential hierarchy in carbon flowMOLECULAR MICROBIOLOGY, Issue 3 2006Robert P. Shivers Summary Conversion of pyruvate to acetate via the phosphotransacetylase-acetate kinase pathway generates ATP and is a major overflow pathway under conditions of carbon and nitrogen excess. In Bacillus subtilis, this pathway is positively regulated by CcpA, a global regulator of carbon metabolism genes. Transcription of the acetate kinase gene (ackA) proved to be activated as well by a second global regulatory protein, CodY. Expression of an ackA,lacZ fusion was reduced in a codY mutant strain. CodY was found to bind in vitro to two sites in the ackA promoter region and to stimulate ackA transcription in a run-off transcription assay. This is the first known case of direct positive regulation by CodY. CodY and CcpA were found to bind to neighbouring sites and their effects were additive both in vivo and in vitro. Surprisingly, positive regulation by CodY, unlike repression, responded primarily to only one type of effector molecule. That is, branched-chain amino acids (BCAAs) served as more potent co-activators of CodY-dependent ackA transcription than did GTP. Given the roles of CcpA and CodY in regulating genes whose products determine the metabolic fate of pyruvate, these two proteins may act together to mediate a hierarchical conversion of pyruvate to its many potential products. [source] | |||||||||||||||||||||||||||||||||||||