- Volume 154, Issue 11, 2008
Volume 154, Issue 11, 2008
- Mini-Review
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A model of efficiency: stress tolerance by Streptococcus mutans
More LessThe complete genome sequence of Streptococcus mutans, a bacterial pathogen commonly associated with human dental caries, was published in 2002. The streamlined genome (2.03 Mb) revealed an organism that is well adapted to its obligately host-associated existence in multispecies biofilms on tooth surfaces: a dynamic environment that undergoes rapid and substantial fluctuations. However, S. mutans lacks many of the sensing systems and alternative sigma factors that bacteria often use to coordinate gene expression in response to stress and changes in their environment. Over the past 7 years, functional genomics and proteomics have enhanced our understanding of how S. mutans has integrated the stress regulon and global transcriptional regulators to coordinate responses to environmental fluctuations with modulation of virulence in a way that ensures persistence in the oral cavity and capitalizes on conditions that are favourable for the development of dental caries. Here, we highlight advances in dissection of the stress regulon of S. mutans and its intimate interrelationship with pathogenesis.
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- Microbiology Comment
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- Biochemistry And Molecular Biology
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The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072)
An endophytic fungus, Gliocladium roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed ‘myco-diesel’. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.
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The msiK gene, encoding the ATP-hydrolysing component of N,N′-diacetylchitobiose ABC transporters, is essential for induction of chitinase production in Streptomyces coelicolor A3(2)
More LessThe dasABC genes encode an ATP-binding cassette (ABC) transporter, which is one of the uptake systems for N,N′-diacetylchitobiose [(GlcNAc)2] in Streptomyces coelicolor A3(2), although the gene encoding the ABC subunit that provides ATP hydrolysis for DasABC has not been identified. In this study, we disrupted the sequence that is highly homologous to the msiK gene, the product of which is an ABC subunit assisting several ABC permeases in other Streptomyces species. Disruption of msiK severely affected the ability of S. coelicolor A3(2) to utilize maltose, cellobiose, starch, cellulose, chitin and chitosan, but not glucose. The msiK null mutant lacked (GlcNAc)2-uptake activity, but GlcNAc transport activity was unaffected. The data indicated that msiK is essential for (GlcNAc)2 uptake, which in S. coelicolor A3(2) is governed by ABC transporters including the DasABC–MsiK system, in contrast to Escherichia coli and Serratia marcescens, in which (GlcNAc)2 uptake is mediated by the phosphotransferase system. Interestingly, the induction of chitinase production by (GlcNAc)2 or chitin was absent in the msiK null mutant, unlike in the parent strain M145. The defect in chitinase gene induction was rescued by expressing the His-tagged MsiK protein under the control of the putative native promoter on a multicopy plasmid. The data suggest that uptake of (GlcNAc)2 is necessary for induction of chitinase production. The msiK gene was constitutively transcribed, whereas the transcription of dasA [(GlcNAc)2-binding protein gene], malE (putative maltose-binding protein gene), cebE1 (putative cellobiose-binding protein gene) and bxlE1 (putative xylobiose-binding protein gene) was induced by their corresponding sugar ligands. This is believed to be the first report to indicate that (GlcNAc)2 uptake mediated by ABC transporters is essential for chitinase production in streptomycetes, which are known to be the main degraders of chitin in soil.
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Determination of the minimum domain II size of Escherichia coli DnaA protein essential for cell viability
More LessThe DnaA protein is the bacterial initiator of replication at a unique chromosomal site, oriC. It is present in all bacterial species and has a conserved structure with four domains. The structures of domains I and III–IV have been solved recently for some bacterial species, and the molecular process leading to the initiation event has been investigated in detail. On the other hand, domain II appears to have no rigid structure and is assumed to be a flexible linker connecting the N-terminal domain I and the C-terminal domains III–IV. It differs significantly in length and amino acid sequence among bacterial species. Whether or not domain II has any function(s) to initiate replication is unknown. The precise borders at both of its ends as well as its essential portions for cell viability are also unknown. In this study, we introduced systematic deletions into the domain II region on the chromosomal dnaA gene of Escherichia coli and examined their effect on cell physiology. Stretches of 30–36 consecutive amino acid residues could be deleted from various portions between the 78th and the 136th residues without affecting cell viability. We propose that domain II of E. coli DnaA is from the 79th to the 135th residues and at least 21–27 residues are required as a spacer to keep domains I and III–IV in the correct positions.
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A functional Campylobacter jejuni maf4 gene results in novel glycoforms on flagellin and altered autoagglutination behaviour
Flagellin of Campylobacter jejuni is extensively modified with (derivatives of) pseudaminic acid. The flagellar glycosylation locus contains several genes with homopolymeric G-tracts prone to slipped-strand mispairing, some of which belong to the maf gene family. We investigated the function of the putative phase-variable maf4 gene of C. jejuni strain 108. A constructed maf4 mutant displayed unaltered flagella assembly and bacterial motility. 2D-PAGE analysis revealed that the flagellin of strain 108 migrated at a more acidic pI than the protein of the Maf4 mutant. MS-MS in combination with high-resolution matrix-assisted laser desorption/ionization Fourier transform ion cyclotron MS (MALDI-FT-ICR-MS) on flagellin-derived glycopeptides showed that the flagellins of the mutant lacked two previously unidentified modifications of pseudaminic acid. These glycoforms carried additional CO2 and C2H2O2 groups, consistent with the more acidic pI of the wild-type flagellin. Phenotypically, the maf4 mutant displayed strongly delayed bacterial autoagglutination. Collectively, our results suggest that the presence of a functional Maf4 expands the flagellin glycan repertoire with novel glycoforms of pseudaminic acid and, in the event of phase variation, alters the population behaviour of C. jejuni.
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Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology
More LessAcinetobacter baumannii forms biofilms on abiotic surfaces, a phenotype that may explain its ability to survive in nosocomial environments and to cause device-related infections in compromised patients. The biofilm proficiency of the 19606 type strain depends on the production of pili, cell-surface appendages assembled via the CsuAB-A-B-C-D-E chaperone–usher secretion system. The screening of a bank of isogenic insertion derivatives led to the identification of a biofilm-deficient derivative in which a transposon insertion disrupted a gene predicted to encode the response regulator of a two-component regulatory system. This gene, which was named bfmR, is required for the expression of the Csu pili chaperone–usher assembly system. This coding region is followed by an ORF encoding a putative sensor kinase that was named bfmS, which plays a less relevant role in biofilm formation when cells are cultured in rich medium. Further examination showed that the bfmR mutant was capable of attaching to abiotic surfaces, although to levels significantly lower than those of the parental strain, when it was cultured in a chemically defined minimal medium. Additionally, the morphology of planktonic cells of this mutant, when grown in minimal medium, was drastically affected, while adherent mutant cells were indistinguishable in shape and size from the parental strain. Together, these results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.
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Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism
More LessCertain amino acids, and cysteine in particular, promptly blocked toxin expression in Clostridium difficile strain VPI 10463 when added to late-exponential-phase peptone-yeast cultures, i.e. prior to normal induction of toxins A and B. Glucose reduced toxin yields by 80-fold, but only when supplemented at inoculation. Forty upregulated C. difficile proteins were identified during maximum toxin expression, and most of these were enzymes involved in energy exchange, e.g. succinate, CO/folate and butyrate metabolism. Transcription of tcdA (toxin operon) and folD (CO/folate operon) was induced by 20- and 10-fold, respectively, and with strikingly similar kinetics between OD 0.8 and 1.2. The sigma factors tcdR and sigH were upregulated simultaneously with tcdA and folD (3.5-fold increase of mRNA level), whereas transcription of tcdC, codY, sigB and sigL showed little or no correlation with that of tcdA and folD. The results suggest a connection between toxin expression, alternative energy metabolism and initial sporulation events in C. difficile.
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Chlamydophila pneumoniae HflX belongs to an uncharacterized family of conserved GTPases and associates with the Escherichia coli 50S large ribosomal subunit
Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [γ-32P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.
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The endonuclease activity of RNase III is required for the regulation of antibiotic production by Streptomyces coelicolor
More LessThe double strand-specific endoRNase RNase III globally regulates the production of antibiotics by Streptomyces coelicolor. We have undertaken studies to determine whether the endoRNase activity of S. coelicolor RNase III or its RNA binding activity is responsible for its regulatory function. We show that an rnc null mutant of S. coelicolor M145 does not produce actinorhodin or undecylprodigiosin. Restoring a wild-type copy of rnc to that mutant also restored antibiotic production. We constructed an rnc point mutant, D70A, in which an aspartic acid residue which is essential for the catalytic activity of RNase III was changed to alanine. The D70A mutation abolished the catalytic activity of the protein but not its ability to bind to RNA substrates. Introduction of a copy of the D70A gene into the rnc null mutant did not restore antibiotic production. This result suggests that the endoRNase activity of RNase III is required for the regulation of antibiotic production in S. coelicolor. We also reconstructed the C120 point mutation that was originally described in 1992. Although that mutation diminished antibiotic production by S. coelicolor, we confirm here that the C120 protein retains some RNase III activity.
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Determination of a transcriptional regulator-like gene involved in biosynthesis of elsinochrome phytotoxin by the citrus scab fungus, Elsinoë fawcettii
More LessElsinochromes are nonhost-selective, light-activated, polyketide-derived toxins produced by many phytopathogenic Elsinoë species. We recently showed that the polyketide synthase-encoding gene EfPKS1 is essential for elsinochrome biosynthesis in the citrus scab fungus Elsinoë fawcettii. Sequence analysis beyond the EfPKS1 gene identified nine putative ORFs: four genes, designated RDT1, TSF1, PRF1 and ECT1, all encode polypeptides likely to have biosynthetic or efflux functions; five additional genes, OXR1 and EfHP1 to EfHP4, encode hypothetical proteins of unknown function. Northern-blot analysis revealed that expression of these genes in E. fawcettii was not completely correlated with accumulation of elsinochromes under nitrogen limitation, alkaline pH or high concentrations of glucose. Targeted disruption of the TSF1 gene, encoding a putative transcriptional activator, yielded fungal mutants unable to produce elsinochromes, and defective in both conidiation and expression of RDT1, EfPKS1, PRF1 and EfHP1, whereas expression of RDT1, TSF1, PRF1 and ECT1 was nearly abolished in EfPKS1-disrupted mutants. By contrast, expression of OXR1, EfHP2 and EfHP3 was not affected by disrupting either EfPKS1 or TSF1. Taken together, the results indicate that in addition to polyketide synthase, the products of TSF1 and other adjacent genes may also play a crucial role in elsinochrome production.
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Functional impact of mutational activation on the Listeria monocytogenes central virulence regulator PrfA
More LessThe transcriptional activator PrfA is required for the expression of virulence factors necessary for Listeria monocytogenes pathogenesis. PrfA is believed to become activated following L. monocytogenes entry into the cytosol of infected host cells, resulting in the induction of target genes whose products are required for bacterial intracellular growth and cell-to-cell spread. Several mutations have been identified that appear to lock PrfA into its highly activated cytosolic form (known as prfA* mutations). In this study PrfA and five PrfA* mutant proteins exhibiting differing degrees of activity were purified and analysed to define the influences of the mutations on distinct aspects of PrfA activity. Based on limited proteolytic digestion, conformational changes were detected for the PrfA* mutant proteins in comparison to wild-type PrfA. For all but one mutant (PrfA Y63C), the DNA binding affinity as measured by electophoretic mobility shift assay appeared to directly correlate with levels of PrfA mutational activation, such that the high-activity mutants exhibited the largest increases in DNA binding affinity and moderately activated mutants exhibited more moderate increases. Surprisingly, the ability of PrfA and PrfA* mutants to form dimers in solution appeared to inversely correlate with levels of PrfA-dependent gene expression. Based on comparisons of protein activity and structural similarities with PrfA family members Crp and CooA, the prfA* mutations modify distinct aspects of PrfA activity that include DNA binding and protein–protein interactions.
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- Biodiversity And Evolution
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Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans
The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. longibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.
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Genetic diversity of the endemic gourmet mushroom Thelephora ganbajun from south-western China
The ectomycorrhizal fungus Thelephora ganbajun is an endemic gourmet mushroom in Yunnan province, south-western China. However, despite its widespread consumer appeal, nutritional value and potential ecological role in natural forests, very little is known about its genetics, diversity and ecology. In this study, we investigated DNA sequence variation at the internal transcribed spacer (ITS) regions among 156 specimens collected from 23 sites of nine regions in Yunnan Province. Our analysis identified a total of 34 ITS haplotypes and these haplotypes were clustered into five distinct phylogenetic groups. The evolutionary divergences among these clades are similar to or greater than many known sister species pairs within the genus Thelephora and the closely related genus Tomentella. Among the 34 ITS haplotypes, 22 were represented by one specimen each and the remaining 12 were each shared by two or more specimens. The most common haplotype contained 68 specimens distributed in 21 of the 23 sites, a result consistent with gene flow among geographical populations. However, analysis of molecular variance (AMOVA) revealed low but significant genetic differentiation among local and regional populations. Interestingly, the Mantel test identified that the extent of genetic differentiation was not significantly correlated with geographical distance. Our study revealed significant genetic divergence within Th. ganbajun and limited but detectable gene flow among geographical populations of this endemic ectomycorrhizal gourmet mushroom.
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- Environmental Microbiology
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Carbon source-dependent modulation of NADP-glutamate dehydrogenases in isophthalate-degrading Pseudomonas aeruginosa strain PP4, Pseudomonas strain PPD and Acinetobacter lwoffii strain ISP4
More LessAcinetobacter lwoffii strain ISP4 metabolizes isophthalate rapidly compared with Pseudomonas aeruginosa strain PP4 and Pseudomonas strain PPD. Isophthalate has been reported to be a potent competitive inhibitor of glutamate dehydrogenase (GDH). Exogenous supplementation of isophthalate with glutamate or α-ketoglutarate at 1 mM concentration caused strains PP4 and PPD to grow faster than in the presence of isophthalate alone; however, no such effect was observed in strain ISP4. When grown on isophthalate, all strains showed activity of NADP-dependent GDH (NADP-GDH), while cells grown on glucose, 2× yeast extract-tryptone broth (2YT) or glutamate showed activities of both NAD-dependent GDH (NAD-GDH) and NADP-GDH. Activity staining, inhibition and thermal stability studies indicated the carbon source-dependent presence of two (GDHI and GDHII), three (GDHA, GDHB and GDHC) and one (GDHP) forms of NADP-GDH in strains PP4, PPD and ISP4, respectively. The results demonstrate the carbon source-dependent modulation of different forms of NADP-GDH in these bacterial strains. This modulation may help the efficient utilization of isophthalate as a carbon source by overcoming the inhibitory effect on GDH.
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Metabolic cooperation of Gordonia sp. strain MTCC 4818 and Arthrobacter sp. strain WY in the utilization of butyl benzyl phthalate: effect of a novel co-culture in the degradation of a mixture of phthalates
More LessDegradation of butyl benzyl phthalate (BBP) by a co-culture of Gordonia sp. strain MTCC 4818 and Arthrobacter sp. strain WY was investigated. In the degradation of BBP by the co-culture, the limitations of the individual species in metabolizing BBP were overcome, leading to the development of a consortium capable of complete utilization of this ester. In the degradation of BBP by the co-culture, the presence of multiple esterases was demonstrated in both species by activity staining of non-denaturing gels, indicating their roles in the degradation process. The esterases were found to be inducible, with unique or broad substrate specificities towards BBP and its monoesters. Moreover, a number of catabolic enzymes other than esterases identified in the metabolism of BBP-degraded intermediates facilitated the co-culture-mediated degradation process. The versatility of the co-culture was further established by the rapid and complete degradation of a mixture of phthalate esters of environmental concern.
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Picocyanobacterial community structure of freshwater lakes and the Baltic Sea revealed by phylogenetic analyses and clade-specific quantitative PCR
More LessPhylogenetic relationships among picocyanobacteria from the Syn/Pro clade sensu Sánchez-Baracaldo et al. (2005) were determined using small subunit (ssu) rDNA sequences from novel culture isolates together with environmental samples from the Baltic Sea and seven freshwater lakes. The picocyanobacterial community comprised members of previously identified clades and of two previously undescribed clades. The number of well-supported clades suggests that freshwater picocyanobacterial communities encompass much greater diversity than is found in marine systems. To allow the quantification of community structure and temporal succession, clade-specific ssu rDNA TaqMan assays were designed and implemented. These assays were used to assess picocyanobacterial community structure in two lakes over an annual cycle in 2003/4, and in a small number of Baltic Sea samples collected in July 2003. In the lake-water samples, picocyanobacteria were found to be scarce during most of the year, with members of each clade reaching their peak abundance over a relatively short period during the summer (June to September), although representatives of the Cyanobium clade also developed an autumn peak extending towards the end of October. All four freshwater clades were present in the Baltic Sea, but their distribution was patchy over relatively short spatial scales. The use of molecular tools for describing and quantifying community structures reveals previously unexplored complexity in the phytoplankton and will facilitate the development of a more sophisticated understanding of community dynamics at the base of the food chains in lakes.
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- Genes And Genomes
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The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines
More LessPurines are degraded via uric acid to yield allantoin. Under anaerobic conditions, allantoin is further degraded via carbamoylphosphate to &SetFont Typeface="11"; to provide a nitrogen source and, under aerobic conditions, to 3-phosphoglycerate via glyoxylate for energy production. In this study, we found that a DNA-binding transcription factor AllR, together with AllS, plays a key role in switching control of two pathways, nitrogen assimilation and energy production. The repressor function of AllR is activated in the presence of allantoin, the common substrate for both pathways, leading to repression of the genes for energy production. On the other hand, when glyoxylate is accumulated, AllR is inactivated for derepression of the pathway for energy production. RutR, the master regulator for pyrimidines and arginine, is also involved in this pathway-switching control.
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Optical mapping and 454 sequencing of Escherichia coli O157 : H7 isolates linked to the US 2006 spinach-associated outbreak
More LessOptical maps for five representative clinical, food-borne and bovine-derived isolates from the 2006 Escherichia coli O157 : H7 outbreak linked to fresh spinach in the United States showed a common set of 14 distinct chromosomal markers that define the outbreak strain. Partial 454 DNA sequencing was used to characterize the optically mapped chromosomal markers. The markers included insertions, deletions, substitutions and a simple single nucleotide polymorphism creating a BamHI site. The Shiga toxin gene profile of the spinach-associated outbreak isolates (stx1− stx2 + stx2c +) correlated with prophage insertions different from those in the prototypical EDL933 and Sakai reference strains (stx1+ stx2 + stx2c −). The prophage occupying the yehV chromosomal position in the spinach-associated outbreak isolates was similar to the stx1+ EDL933 cryptic prophage V, but it lacked the stx1 gene. In EDL933, the stx2 genes are within prophage BP933-W at the wrbA chromosomal locus; this locus was unoccupied in the spinach outbreak isolates. Instead, the stx2 genes were found within a chimeric BP933-W-like prophage with a different integrase, inserted at the argW locus in the outbreak isolates. An extra set of Shiga toxin genes, stx2c, was found in the outbreak isolates within a prophage integrated at the sbcB locus. The optical maps of two additional clinical isolates from the outbreak showed a single, different prophage variation in each, suggesting that changes occurred in the source strain during the course of this widespread, multi-state outbreak.
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The transcription programme of the protein-primed halovirus SH1
More LessSH1 is the only reported isolate of a spherical halovirus, a dominant morphotype in hypersaline lakes. The virus lytically infects the haloarchaeon Haloarcula hispanica, and carries a 30.9 kb linear dsDNA genome that, in a previous study, was proposed to contain 56 protein-coding genes, probably organized into between four and eight operons. In the present study, these predictions were directly tested by determining the orientations and lengths of virus transcripts using systematic RT-PCR and primer extension. Seven major transcripts were observed that together covered most of the genome. Six transcripts were synthesized from early in infection (1 h post-infection; p.i.) onwards, while transcript T6 was only detected late in infection (5–6 h p.i.). No transcripts were detected in the inverted terminal repeat sequences or at the extreme right end of the genome (ORFs 55–56). Start points for the major transcripts were mapped by primer extension and corresponded closely to the 5′ termini determined by RT-PCR. Between 1 and 4 h p.i., transcripts usually terminated not far beyond the end of their last coding ORF, but late in infection, transcripts from the same promoters often terminated at more distal points, resulting in much of the genome being transcribed from both strands. Since many of these transcripts are complementary, RNA–RNA interactions are likely, and may play a role in regulating viral gene expression. Puromycin blockage of post-infection protein synthesis significantly altered the levels of certain virus transcripts, indicating that de novo protein synthesis is essential for the correct regulation of SH1 gene expression.
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- Pathogens And Pathogenicity
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Additive attenuation of virulence and cariogenic potential of Streptococcus mutans by simultaneous inactivation of the ComCDE quorum-sensing system and HK/RR11 two-component regulatory system
More LessThe genome of Streptococcus mutans harbours 13 two-component signal transduction systems (TCSTSs). Of these, a peptide-mediated quorum-sensing system, ComCDE, and the HK/RR11 two-component system are well known to regulate several virulence-associated traits in in vitro experiments, including genetic competence, bacteriocin production, biofilm formation and stress responses. In this study, we investigated the hypothesis that inactivation of ComCDE, HK/RR11 or both systems would attenuate the virulence and cariogenicity of S. mutans. The results showed that simultaneous inactivation of both signal transduction systems additively attenuated S. mutans virulence and cariogenicity, since inactivation of either of these systems alone did not result in the same degree of effect. The double deletion mutant SMcde-hk11 was defective in genetic competence, had a reduced acid production, was unable to grow at pH 5.0 and formed an abnormal biofilm with reduced biomass. Animal studies showed that this mutant had reduced capabilities for oral colonization, succession and initiation of dental caries. A competitive index (CI) analysis using a mixed-infection animal model revealed that all the mutants, particularly SMcde-hk11, had reduced fitness in their ecological niches and were unable to compete with the wild-type strain for persistence in dental biofilms. The evidence from this study suggests that the ComCDE and HK/RR11 signal transduction systems can be considered to be novel targets for the development of strategies in the prevention and treatment of S. mutans infections.
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Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis
A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1–10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n=17) or vaginal (n=17) candidiasis. SAP gene expression was also analysed in Δsap1–3, Δsap4–6, Δefg1 and Δefg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4–6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Δsap1–3, expression was compensated for by increased expression of SAP5, and in Δsap4–6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Δsap1–3 and Δsap4–6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Δefg1/cph1 and Δefg1, but SAP5 expression was only dramatically reduced in Δefg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hypha-independent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1–3 and Sap4–6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.
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Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans
More LessA well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1–SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1–SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1–SAP3 or the SAP4–SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Δ mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p–Sap6p are not required for invasion of RHE by C. albicans.
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The Candida albicans phosphatase Inp51p interacts with the EH domain protein Irs4p, regulates phosphatidylinositol-4,5-bisphosphate levels and influences hyphal formation, the cell integrity pathway and virulence
We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P2 levels and the cell integrity pathway, and that PI(4,5)P2 homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.
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Dark brown is the more virulent of the switch phenotypes of Candida glabrata
Candida glabrata undergoes reversible, high-frequency core switching between phenotypes that include dark brown (DB), light brown (LB) and white (Wh). These phenotypes in turn can switch to the irregular wrinkle (IWr) phenotype. Natural isolates, however, express predominantly the DB phenotype, leading to the hypothesis that it has a colonization advantage over the other switch phenotypes. Using the mouse model of systemic infection, results are presented which support this hypothesis. DB has an advantage over other switch phenotypes in colonizing the two major target organs in the mouse model, the spleen and liver. A time-course study reveals that colonization of the major target organs occurs very rapidly (within 2 h) after host injection, and that the DB advantage for spleen and liver colonization is immediate. The DB advantage is maintained during clearing from spleen, liver and kidneys, and during delayed transient brain colonization. These results demonstrate that DB has a colonization advantage over other switch phenotypes, and that the switch phenotype expressed by a colonizing population therefore plays a fundamental role in virulence. It is therefore essential that switching be considered in both in vivo and in vitro studies of C. glabrata virulence.
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Modification of Borrelia burgdorferi to overproduce OspA or VlsE alters its infectious behaviour
More LessThe surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID50, but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID50 increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.
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A comparative study of host response to three Mycobacterium tuberculosis PE_PGRS proteins
More LessThree Mycobacterium tuberculosis proteins, PE_PGRS 16 (Rv0977), PE_PGRS 26 (Rv1441c) and PE_PGRS 33 (Rv1818c), were expressed in Mycobacterium smegmatis and used to investigate the host response to members of this unique protein family. Following infection of macrophages with the recombinant M. smegmatis (Ms) strains, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 were significantly more persistent (4.4 and 4.2 log c.f.u.) compared with Ms-PE_PGRS 16 (3.4 log c.f.u.) at day 6. Similarly, after infection of mice, Ms-PE_PGRS 33 and Ms-PE_PGRS 26 persisted at significantly higher levels in the spleen (3.5 and 3.2 log c.f.u.) and liver (3 and 2.6 log c.f.u.) compared with Ms-PE_PGRS 16 in the spleen (2 log c.f.u.) and in the liver (1 log c.f.u.) at day 10. Increased persistence of Ms-PE_PGRS 33 and Ms-PE_PGRS 26 was associated with cell death and increased release of lactate dehydrogenase in macrophage cultures as well as increased levels of IL-10 and, in contrast, lower levels of IL-12 and NO both in vitro and in mouse splenocytes. Conversely, poor survival of Ms-PE_PGRS 16 was associated both in macrophage cultures and in vivo with higher levels of NO and IL-12. All three PE_PGRS proteins were found to be cell-surface antigens, but immunization of mice with these PE_PGRS antigens as DNA vaccines showed no protection in a TB aerosol challenge model. In general, the results suggest that variable expression of different PE_PGRS proteins within host cells can affect either the fate of the mycobacterial pathogen or that of the host during infection and point to the importance of studying the expression and function of individual members of the PE_PGRS gene family of M. tuberculosis.
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agr RNAIII divergently regulates glucose-induced biofilm formation in clinical isolates of Staphylococcus aureus
Staphylococcus aureus is an important nosocomial and community-acquired pathogen. Hospital infections are frequently complicated by the ability of bacteria to form biofilms on different surfaces. The development of bacterial films on medical indwelling devices, such as prostheses, often requires surgical procedures to remove the contaminated implant. Indeed, biofilm formation on central endovenous catheters is a major cause of primary bacteraemia in hospitals. The modulation of virulence factors in S. aureus is orchestrated by a number of global regulators including agr RNAIII. To improve our understanding of the role of the agr quorum-sensing system in biofilm formation by S. aureus, we constructed a number of agr-null mutants, derived from contemporary clinical isolates. Analysis of these mutants indicates that agr has a significant impact on biofilm development for most of the isolates tested. Our data show that RNAIII can control both biofilm formation and accumulation. The agr effect included both up- and downregulation of biofilms, even for isolates within the same lineage, corroborating the hypothesis that the mechanisms involved in S. aureus biofilms are complex and probably multifactorial.
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Salmonella virulence factor SpiC is involved in expression of flagellin protein and mediates activation of the signal transduction pathways in macrophages
More LessSpiC is a virulence factor encoded within Salmonella pathogenicity island 2 (SPI-2). We have previously reported that infection of macrophages with Salmonella enterica serovar Typhimurium results in the SPI-2-dependent activation of the mitogen-activated protein kinase (MAPK) signalling pathways, leading to the expression of suppressor of cytokine signalling (SOCS)-3, which is involved in the inhibition of cytokine signalling. Here, we investigated the mechanism by which SpiC mediates the activation of signal transduction pathways in macrophages. Proteomic analysis showed that the level of FliC protein, a component of the flagellar filaments, was lower in the culture supernatant of a spiC mutant than in the supernatant from wild-type Salmonella. Furthermore, quantitative real-time RT-PCR showed that this mutant had a much lower level of fliC mRNA, indicating that SpiC regulates the transcription of FliC. We also found that the level of SOCS-3 in J774 macrophages was lower when they were infected with the fliC mutant than with wild-type Salmonella. FliC participated in the activation of MAPK pathways in Salmonella-infected macrophages, leading to the upregulation of SOCS-3 expression. Collectively, these results indicate that SpiC is involved in the expression of FliC, which plays a significant role in the SPI-2-dependent activation of MAPK pathways in Salmonella-infected macrophages.
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The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells
The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode ∼1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 °C and 37 °C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.
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Antibacterial activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis
More LessCyt1Aa is a δ-endotoxin protein that is produced by Bacillus thuringiensis subsp. israelensis. It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23–24 kDa and a threefold increase in activity. In this research, Cyt1Aa crystals were purified from B. thuringiensis IPS78/11 harbouring the expression vector pHT-cyAp20. The activity of the activated form of Cyt1Aa (23–24 kDa) was examined on a pathogenic strain of the Gram-negative Escherichia coli and the Gram-positive species Staphylococcus aureus. The Cyt1Aa minimal inhibitory concentration for E. coli and S. aureus was 1.25 and 5 μg ml−1, respectively. Cyt1Aa was found to be bactericidal for E. coli, whereas it was bacteriostatic for S. aureus. Furthermore, Cyt1Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole δ-endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1Aa has a bactericidal effect upon Gram-negative bacteria.
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Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms
More LessSurvival strategies exhibited over 4 years by Ralstonia solanacearum phylotype (ph) II biovar (bv) 2 in environmental water microcosms were examined. The bacterium is a devastating phytopathogen whose ph II bv 2 causes bacterial wilt in solanaceous crops and ornamental plants. Outbreaks of the disease may originate from dissemination of the pathogen in watercourses, where it has to cope with prolonged nutrient limitation. To ascertain the effect of long-term starvation on survival and pathogenicity of R. solanacearum in natural water microcosms, survival experiments were conducted. Microcosms were prepared from different sterile river water samples, inoculated separately with two European strains of ph II at 106 c.f.u. ml−1 and maintained at 24 °C for 4 years. In all assayed waters, starved R. solanacearum remained in a non-growing but culturable state during the first year, maintaining approximately the initial numbers. Thereafter, part of the population of R. solanacearum progressively lost the ability to form colonies, and non-culturable but metabolically active cells appeared. During the whole period, the bacterium remained pathogenic on host plants and underwent a transition from typical bacilli to small cocci which tended to aggregate. Some starved R. solanacearum cells filamented and formed buds. Starvation response, viable but non-culturable state, morphological changes and aggregation have not previously been reported for this pathogen as survival mechanisms induced in oligotrophic conditions. The potential existence of long-starved pathogenic cells in environmental waters may raise new concerns about the epidemiology of bacterial wilt disease.
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- Physiology
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l-Cysteine is required for induced antibiotic resistance in actively swarming Salmonella enterica serovar Typhimurium
More LessSwarm-cell differentiation in Salmonella enterica serovar Typhimurium (S. typhimurium) results in a biosynthetic mode of growth, despite growing on a rich medium, and cells that have elevated antibiotic resistance. These phenotypes are not a prerequisite for swarm motility. By blocking the switch to anabolic growth using amino acid auxotrophs and screening for the presence of elevated antibiotic resistance in the swarm state, we found that cysteine biosynthesis is crucial for complete swarm-cell differentiation. Mutants were made in each cys biosynthetic operon and all had decreased antibiotic resistance in the swarm state, while swim-cell resistance remained the same as that of wild-type cells. This swarm-state-specific decreased resistance in Δcys strains could be restored to wild-type levels by the addition of cysteine to swarm medium. Two regulatory mutants, ΔcysB and ΔcysE, failed to swarm unless cysteine was supplemented to the medium. We show that all CysB-responsive operons involved in cysteine biosynthesis are upregulated in the swarm state, even though swarm cells are cultivated on a medium that represses cysteine biosynthesis in the swim state. While swarm medium has sufficient cysteine for growth of S. typhimurium, it does not contain enough for swarm-cell differentiation. We hypothesize that in these cells, the additional cysteine requirement is for use in pathways not directly related to cell growth.
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The crucial role of mitochondrial regulation in adaptive aluminium resistance in Rhodotorula glutinis
Rhodotorula glutinis IFO1125 was found to acquire increased aluminium (Al) resistance from 50 μM to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. To investigate the mechanism underlying this novel phenomenon, wild-type and Al-resistant cells were compared. Neither cell type accumulated the free form of Al (Al3+) added to the medium. Transmission electron microscopic analyses revealed a greater number of mitochondria in resistant cells. The formation of small mitochondria with simplified cristae structures was observed in the wild-type strain grown in the presence of Al and in resistant cells grown in the absence of Al. Addition of Al to cells resulted in high mitochondrial membrane potential and concomitant generation of reactive oxygen species (ROS). Exposure to Al also resulted in elevated levels of oxidized proteins and oxidized lipids. Addition of the antioxidants α-tocopherol and ascorbic acid alleviated the Al toxicity, suggesting that ROS generation is the main cause of Al toxicity. Differential display analysis indicated upregulation of mitochondrial genes in the resistant cells. Resistant cells were found to have 2.5- to 3-fold more mitochondrial DNA (mtDNA) than the wild-type strain. Analysis of tricarboxylic acid cycle and respiratory-chain enzyme activities in wild-type and resistant cells revealed significantly reduced cytochrome c oxidase activity and resultant high ROS production in the latter cells. Taken together, these data suggest that the adaptive increased resistance to Al stress in resistant cells resulted from an increased number of mitochondria and increased mtDNA content, as a compensatory response to reduced respiratory activity caused by a deficiency in complex IV function.
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The heterotrimeric Gα protein Pga1 regulates biosynthesis of penicillin, chrysogenin and roquefortine in Penicillium chrysogenum
We have studied the role of the pga1 gene of Penicillium chrysogenum, encoding the alpha subunit of a heterotrimeric G protein, in secondary metabolite production. The dominant activating pga1G42R mutation caused an increase in the production of the three secondary metabolites penicillin, the yellow pigment chrysogenin and the mycotoxin roquefortine, whereas the dominant inactivating pga1G203R allele and the deletion of the pga1 gene resulted in a decrease of the amount of produced penicillin and roquefortine. Chrysogenin is produced in solid medium as a yellow pigment, and its biosynthesis is clearly enhanced by the presence of the dominant activating pga1G42R allele. Roquefortine is produced associated with mycelium during the first 3 days in submerged cultures, and is released to the medium afterwards; dominant activating and inactivating pga1 mutations result in upregulation and downregulation of roquefortine biosynthesis recpectively. Pga1 regulates penicillin biosynthesis by controlling expression of the penicillin biosynthetic genes; the three genes pcbAB, pcbC and penDE showed elevated transcript levels in transformants expressing the pga1G42R allele, whereas in transformants with the inactivating pga1G203R allele and in the pga1-deleted mutant their transcript levels were lower than those in the parental strains. Increase of intracellular cAMP levels had no effect on penicillin production. In summary, the dominant activating pga1G42R allele upregulates the biosynthesis of three secondary metabolites in Penicillium chrysogenum to a different extent.
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