- Volume 158, Issue 1, 2012
Volume 158, Issue 1, 2012
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Trichoderma: sensing the environment for survival and dispersal
More LessSpecies belonging to the genus Trichoderma are free-living fungi common in soil and root ecosystems, and have a broad range of uses in industry and agricultural biotechnology. Some species of the genus are widely used biocontrol agents, and their success is in part due to mycoparasitism, a lifestyle in which one fungus is parasitic on another. In addition Trichoderma species have been found to elicit plant defence responses and to stimulate plant growth. In order to survive and spread, Trichoderma switches from vegetative to reproductive development, and has evolved with several sophisticated molecular mechanisms to this end. Asexual development (conidiation) is induced by light and mechanical injury, although the effects of these inducers are influenced by environmental conditions, such as nutrient status and pH. A current appreciation of the links between the molecular participants is presented in this review. The photoreceptor complex BLR-1/BLR-2, ENVOY, VELVET, and NADPH oxidases have been suggested as key participants in this process. In concert with these elements, conserved signalling pathways, such as those involving heterotrimeric G proteins, mitogen-activated protein kinases (MAPKs) and cAMP-dependent protein kinase A (cAMP-PKA) are involved in this molecular orchestration. Finally, recent comparative and functional genomics analyses allow a comparison of the machinery involved in conidiophore development in model systems with that present in Trichoderma and a model to be proposed for the key factors involved in the development of these structures.
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Plant-beneficial effects of Trichoderma and of its genes
More LessTrichoderma (teleomorph Hypocrea) is a fungal genus found in many ecosystems. Trichoderma spp. can reduce the severity of plant diseases by inhibiting plant pathogens in the soil through their highly potent antagonistic and mycoparasitic activity. Moreover, as revealed by research in recent decades, some Trichoderma strains can interact directly with roots, increasing plant growth potential, resistance to disease and tolerance to abiotic stresses. This mini-review summarizes the main findings concerning the Trichoderma–plant interaction, the molecular dialogue between the two organisms, and the dramatic changes induced by the beneficial fungus in the plant. Efforts to enhance plant resistance and tolerance to a broad range of stresses by expressing Trichoderma genes in the plant genome are also addressed.
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Self versus non-self: fungal cell wall degradation in Trichoderma
More LessLysis of the prey’s cell wall is one of the key steps during mycoparasitism. Genome analysis of two mycoparasitic Trichoderma species, T. atroviride and T. virens, revealed an expanded arsenal of genes encoding enzymes potentially involved in cell wall hydrolysis. Glycoside hydrolase family 18, which contains all fungal chitinases, is the largest family of carbohydrate-active enzymes in mycoparasitic Trichoderma species. However, in addition to their aggressive functions during mycoparasitism, the roles of chitinases and other cell wall degrading enzymes also include remodelling and recycling of the fungus’s own cell wall. In this review we discuss current knowledge about fungal cell wall degrading enzymes in Trichoderma and how the fungus distinguishes between self- and non-self fungal cell wall degradation. In the past few years, the chitinolytic enzyme machinery of Trichoderma has been used as a model system to address this question. Gene expression profiles of most investigated chitinases indicate an overlap of functions of the respective enzymes and an involvement in both self- and non-self fungal cell wall degradation. Similar sets of enzymes appear to be involved in mycoparasitism, exogenous chitin decomposition and recycling of the fungus’s own cell wall. Thus, we hypothesize that the regulation of self and non-self fungal cell wall degradation is not due to a speciation of individual chitinases. Rather, we hypothesize that it is regulated by substrate accessibility due to cell wall protection in healthy hyphae vs deprotection during mycoparasitic attack, hyphal ageing and autolysis.
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Secondary metabolism in Trichoderma – a genomic perspective
More LessTrichoderma spp. are a rich source of secondary metabolites (SMs). The recent publication of the genome sequences of three Trichoderma spp. has revealed a vast repertoire of genes putatively involved in the biosynthesis of SMs, such as non-ribosomal peptides, polyketides, terpenoids and pyrones. Interestingly, the genomes of the mycoparasitic species Trichoderma virens and Trichoderma atroviride are enriched in secondary metabolism-related genes compared with the biomass-degrading Trichoderma reesei: 18 and 18 polyketide synthases compared with 11; 28 and 16 non-ribosomal peptide synthetases compared with 10, respectively. All three species produce a special class of non-ribosomally synthesized peptides known as peptaibols, containing non-proteinogenic amino acids (particularly α-aminoisobutyric acid). In common with other filamentous ascomycetes, Trichoderma spp. may require siderophores (also produced by non-ribosomal peptide synthetases) to grow in iron-poor conditions and to compete with their hosts for available iron. Two generalizations can be made about fungal SM genes: they are often found in clusters, and many are not expressed under standard laboratory conditions. This has made it difficult to identify the compounds. Trichoderma, in particular, interacts with other microbes in the soil and with plant roots in the rhizosphere. A detailed metabolomic–genomic study would eventually unravel the roles of many of these SMs in natural ecosystems. Novel genetic tools developed recently, combined with biological understanding of the function of SMs as toxins or signals, should lead to ‘awakening’ of these ‘silent’ clusters. Knowledge of the SM repertoire should precede application of Trichoderma strains for biocontrol: some metabolites could be toxic to plants and their consumers, and thus should be avoided. Others could be beneficial, antagonizing pathogens or inducing resistance in crop plants.
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The cargo and the transport system: secreted proteins and protein secretion in Trichoderma reesei (Hypocrea jecorina)
More LessTrichoderma reesei (Hypocrea jecorina) is an efficient cell factory for protein production that is exploited by the enzyme industry. Yields of over 100 g secreted protein l−1 from industrial fermentations have been reported. In this review we discuss the spectrum of proteins secreted by T. reesei and the studies carried out on its protein secretion system. The major enzymes secreted by T. reesei under production conditions are those degrading plant polysaccharides, the most dominant ones being the major cellulases, as demonstrated by the 2D gel analysis of the secretome. According to genome analysis, T. reesei has fewer genes encoding enzymes involved in plant biomass degradation compared with other fungi with sequenced genomes. We also discuss other T. reesei secreted enzymes and proteins that have been studied, such as proteases, laccase, tyrosinase and hydrophobins. Investigation of the T. reesei secretion pathway has included molecular characterization of the pathway components functioning at different stages of the secretion process as well as analysis of the stress responses caused by impaired folding or trafficking in the pathway or by expression of heterologous proteins. Studies on the transcriptional regulation of the secretory pathway have revealed similarities, but also interesting differences, with other organisms, such as a different induction mechanism of the unfolded protein response and the repression of genes encoding secreted proteins under secretion stress conditions.
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Trichoderma reesei RUT-C30 – thirty years of strain improvement
More LessThe hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic- and proteomic-based research on the RUT-C30 strain published over the last 30 years.
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Taxon-specific metagenomics of Trichoderma reveals a narrow community of opportunistic species that regulate each other’s development
More LessIn this paper, we report on the in situ diversity of the mycotrophic fungus Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya) revealed by a taxon-specific metagenomic approach. We designed a set of genus-specific internal transcribed spacer (ITS)1 and ITS2 rRNA primers and constructed a clone library containing 411 molecular operational taxonomic units (MOTUs). The overall species composition in the soil of the two distinct ecosystems in the Danube floodplain consisted of 15 known species and two potentially novel taxa. The latter taxa accounted for only 1.5 % of all MOTUs, suggesting that almost no hidden or uncultivable Hypocrea/Trichoderma species are present at least in these temperate forest soils. The species were unevenly distributed in vertical soil profiles although no universal factors controlling the distribution of all of them (chemical soil properties, vegetation type and affinity to rhizosphere) were revealed. In vitro experiments simulating infrageneric interactions between the pairs of species that were detected in the same soil horizon showed a broad spectrum of reactions from very strong competition over neutral coexistence to the pronounced synergism. Our data suggest that only a relatively small portion of Hypocrea/Trichoderma species is adapted to soil as a habitat and that the interaction between these species should be considered in a screening for Hypocrea/Trichoderma as an agent(s) of biological control of pests.
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Saprotrophic competitiveness and biocontrol fitness of a genetically modified strain of the plant-growth-promoting fungus Trichoderma hamatum GD12
Trichoderma species are ubiquitous soil fungi that hold enormous potential for the development of credible alternatives to agrochemicals and synthetic fertilizers in sustainable crop production. In this paper, we show that substantial improvements in plant productivity can be met by genetic modification of a plant-growth-promoting and biocontrol strain of Trichoderma hamatum, but that these improvements are obtained in the absence of disease pressure only. Using a quantitative monoclonal antibody-based ELISA, we show that an N-acetyl-β-d-glucosaminidase-deficient mutant of T. hamatum, generated by insertional mutagenesis of the corresponding gene, has impaired saprotrophic competitiveness during antagonistic interactions with Rhizoctonia solani in soil. Furthermore, its fitness as a biocontrol agent of the pre-emergence damping-off pathogen Sclerotinia sclerotiorum is significantly reduced, and its ability to promote plant growth is constrained by the presence of both pathogens. This work shows that while gains in T. hamatum-mediated plant-growth-promotion can be met through genetic manipulation of a single beneficial trait, such a modification has negative impacts on other aspects of its biology and ecology that contribute to its success as a saprotrophic competitor and antagonist of soil-borne pathogens. The work has important implications for fungal morphogenesis, demonstrating a clear link between hyphal architecture and secretory potential. Furthermore, it highlights the need for a holistic approach to the development of genetically modified Trichoderma strains for use as crop stimulants and biocontrol agents in plant agriculture.
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Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium
More LessFusarium head blight (FHB) is a re-emerging wheat disease that causes extensive damage through direct losses in yield and quality due to the presence of damaged Fusarium kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists, in order to reduce pathogen inoculum of FHB, holds considerable promise. Ten Trichoderma isolates, previously selected for their ability to grow in the presence of DON, were preliminarily investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic strains in plate confrontation assays. The three Trichoderma isolates showing antibiosis and mycoparasitism were evaluated for their capacity to inhibit DON production by F. graminearum and F. culmorum on two natural substrates. The expression of some chitinase-encoding genes by the two best resulting Trichoderma strains, during interaction with F. culmorum and F. graminearum, was monitored. All investigated genes from chitinase subgroups A, B and the new subgroup C responded to mycoparasitic conditions and were upregulated before contact and/or when in contact with the host. T. gamsii 6085, the best antagonist, was finally used in a competition test against F. culmorum and F. graminearum on natural substrates, using a qPCR approach to evaluate its effect on the pathogen’s growth and DON production in haulms and rice. This test confirmed the ability of T. gamsii 6085 to antagonize the pathogens on rice. On wheat haulms, an extreme oligotrophic environment, T. gamsii 6085 seemed to develop very poorly and the growth of both the pathogens was unaffected by the presence of the antagonist.
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The seven-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host
More LessMycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay.
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Comparative study of Trichoderma gene expression in interactions with tomato plants using high-density oligonucleotide microarrays
More LessTrichoderma spp. are widely used as biopesticides and biofertilizers to control diseases and to promote positive physiological responses in plants. In vitro and in vivo assays with Trichoderma harzianum CECT 2413 (T34), Trichoderma virens Gv29-8 (T87) and Trichoderma hamatum IMI 224801 (T7) revealed that these strains affected the growth and development of lateral roots in tomato plants in different ways. The early expression profiles of these Trichoderma strains were studied after 20 h of incubation in the presence of tomato plants, using a high-density oligonucleotide (HDO) microarray, and compared to the profiles in the absence of plants. Out of the total 34 138 Trichoderma probe sets deposited on the microarray, 1077 (3.15 %) showed a significant change of at least 2-fold in expression in the presence of tomato plants. The numbers of probe sets identified in the individual Trichoderma strains were 593 in T. harzianum T34, 336 in T. virens T87 and 94 in T. hamatum T7. Carbohydrate metabolism – the chitin degradation enzymes N-acetylglucosamine-6-phosphate deacetylase, glucosamine-6-phosphate deaminase and chitinase – was the most significantly overrepresented process commonly observed in the three Trichoderma strains in early interactions with tomato plants. Strains T7 and T34, which had similar positive effects on plant development in biological assays, showed a significantly overrepresented hexokinase activity in interaction with tomato. In addition, genes encoding a 40S ribosomal protein and a P23 tumour protein were altered in both these strains.
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The qid74 gene from Trichoderma harzianum has a role in root architecture and plant biofertilization
More LessThe Trichoderma harzianum qid74 gene encodes a cysteine-rich cell wall protein that has an important role in adherence to hydrophobic surfaces and cellular protection; this gene was upregulated in Trichoderma high-density oligonucleotide (HDO) microarrays in interaction with tomato roots. Using a collection of qid74-overexpressing and disrupted mutants the role of this gene in cucumber and tomato root architecture was analysed in hydroponic and soil systems under greenhouse conditions. No significant differences were found in the pattern of root colonization and the length of primary roots of cucumber or tomato plants inoculated by T. harzianum CECT 2413 wild-type (wt) strain or any of the qid74 transformants. However, compared to the wt treatment, lateral roots were significantly longer in plants inoculated with the overexpressing transformants, and shorter in those treated with the disruptant strains. Microscopic observations revealed more and longer secondary root hairs in cucumber plants treated with the qid74-overexpressing mutants and fewer and shorter hairs in roots treated with qid74-disrupted transformants, compared to those observed in plants inoculated with the wt strain. qid74-induced modifications in root architecture increased the total absorptive surface, facilitating nutrient uptake and translocation of nutrients in the shoots, resulting in increased plant biomass through an efficient use of NPK and micronutrients.
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Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to Pseudomonas syringae in Arabidopsis thaliana
More LessIn the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.
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Phylogenomic analysis of polyketide synthase-encoding genes in Trichoderma
More LessMembers of the economically important ascomycete genus Trichoderma are ubiquitously distributed around the world. The mycoparasitic lifestyle and plant defence-inducing interactions of Trichoderma spp. make them ideal biocontrol agents. Of the Trichoderma enzymes that produce secondary metabolites, some of which likely play important roles in biocontrol processes, polyketide synthase (PKSs) have garnered less attention than non-ribosomal peptide synthetases such as those that produce peptaibols. We have taken a phylogenomic approach to study the PKS repertoire encoded in the genomes of Trichoderma reesei, Trichoderma atroviride and Trichoderma virens. Our analysis lays a foundation for future research related to PKSs within the genus Trichoderma and in other filamentous fungi.
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Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize
Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma–plant interactions resulting in induction of defence responses.
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Antimicrobial peptaibols from Trichoderma pseudokoningii induce programmed cell death in plant fungal pathogens
Antibiosis is one of the widespread strategies used by Trichoderma spp. against plant fungal pathogens, the mechanism of which, however, remains poorly understood. Peptaibols are a large family of antimicrobial peptides produced by Trichoderma spp. Our previous study showed that trichokonins, a type of peptaibol from Trichoderma pseudokoningii SMF2, exhibited antibiotic activities against plant fungal pathogens. In this study, we first demonstrated that trichokonin VI (TK VI) induced extensive apoptotic programmed cell death in plant fungal pathogens. For a deeper insight into the apoptotic mechanism involved in the action of TK VI, Fusarium oxysporum was used as a model. Cells of F. oxysporum treated with TK VI showed apoptotic hallmarks, such as exposure of phosphatidylserine, the appearance of reactive oxygen species and fragmentation of nuclear DNA. Moreover, TK VI-treated cells exhibited an accumulation of cytoplasmic vacuoles with loss of the mitochondrial transmembrane potential, and this process was independent of metacaspases. Therefore, TK VI induces metacaspase-independent apoptotic cell death in F. oxysporum. This represents what is believed to be the first report to reveal the antibiotic mechanism of peptaibols against plant fungal pathogens.
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- Review
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PII signal transduction proteins: pivotal players in post-translational control of nitrogenase activity
The fixation of atmospheric nitrogen by the prokaryotic enzyme nitrogenase is an energy- expensive process and consequently it is tightly regulated at a variety of levels. In many diazotrophs this includes post-translational regulation of the enzyme’s activity, which has been reported in both bacteria and archaea. The best understood response is the short-term inactivation of nitrogenase in response to a transient rise in ammonium levels in the environment. A number of proteobacteria species effect this regulation through reversible ADP-ribosylation of the enzyme, but other prokaryotes have evolved different mechanisms. Here we review current knowledge of post-translational control of nitrogenase and show that, for the response to ammonium, the PII signal transduction proteins act as key players.
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- Cell and Molecular Biology of Microbes
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The global impact of the delta subunit RpoE of the RNA polymerase on the proteome of Streptococcus mutans
More LessTranscriptional specificity in low-G+C Gram-positive bacteria is maintained by RpoE, the delta subunit of the RNA polymerase. Here, we studied the effect of RpoE at the proteome level in the human dental pathogen Streptococcus mutans by comparing the ΔrpoE mutant with the wild-type under five conditions: (0) exponential growth, (1) early stationary phase, (2) acid stress, (3) oxidative stress, and (4) combined acid and oxidative stress. A total of 280 cellular protein spots were reproducibly detected, of which 97 differentially expressed protein spots were identified by MALDI-TOF MS. Lack of RpoE caused downregulation of proteins for carbohydrate metabolism and energy production, including phosphoglucomutase (PGM), the phosphopentomutase DeoB and the pyruvate formate-lyase Pfl. The ΔrpoE mutant had extensive changes in the abundance of proteins involved in acid and oxidative tolerance and protein turnover, and of chaperones, at exponential phase in the absence of stress, suggesting a potential internal stress. In addition, the mutant had reduced amounts of proteins for adaptation responses, e.g. the multiple sugar transport and metabolism enzymes required for entering early stationary phase, and the proteins for stress-defence mechanisms and glycolysis under oxidative stress. Comparison of the proteome data with the corresponding transcriptome data suggested that the effects were the result of altered transcriptional and post-transcriptional regulation. The data are consistent with the reduced transcriptional specificity of the RNA polymerase in the ΔrpoE mutant, and suggest a general impact, but not a specific regulatory role, of RpoE in stress adaptation.
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Stringent response mutants of Pseudomonas chlororaphis PA23 exhibit enhanced antifungal activity against Sclerotinia sclerotiorum in vitro
More LessThe stringent response (SR) is a regulatory mechanism that enables bacteria to adapt to nutrient stress through the production of the alarmone (p)ppGpp. The aim of the current study was to understand how the SR affects the antifungal (AF) activity of Pseudomonas chlororaphis PA23. Two SR mutants were generated, PA23relA and PA23relAspoT, that no longer produced (p)ppGpp. Both mutants exhibited increased inhibition of Sclerotinia sclerotiorum in vitro and elevated pyrrolnitrin (PRN), lipase and protease production. Phenazine (PHZ) levels, on the other hand, remained unchanged. Through transcriptional fusion analysis we discovered that prnA–lacZ (PRN) activity was increased in the SR mutants, whereas phzA–lacZ (PHZ) activity was equal to that of the wild-type. We also examined how the sigma factor RpoS impacts PA23-mediated antagonism. Similar to the SR mutants, an rpoS mutant of PA23, called PA23rpoS, exhibited enhanced AF activity in vitro and increased expression of PRN, protease and lipase. However, PHZ production and expression of phzA–lacZ were dramatically reduced. Consistent with what has been reported for other bacteria, the SR exerted positive control over rpoS expression. In addition, providing rpoS in trans restored the SR phenotype to that of the wild-type. Collectively, our findings indicate that this global stress response impacts production of PA23 AF compounds via regulation of rpoS transcription and has an overall negative influence on S. sclerotiorum antagonism.
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- Genes and Genomes
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Alternative routes of acetyl-CoA synthesis identified by comparative genomic analysis: involvement in the lipid production of oleaginous yeast and fungi
More LessFor a bio-based economy, microbial lipids offer a potential solution as alternative feedstocks in the oleochemical industry. The existing genome data for the promising strains, oleaginous yeasts and fungi, allowed us to investigate candidate orthologous sequences that participate in their oleaginicity. Comparative genome analysis of the non-oleaginous (Saccharomyces cerevisiae, Candida albicans and Ashbya gossypii) and oleaginous strains (Yarrowia lipolytica, Rhizopus oryzae, Aspergillus oryzae and Mucor circinelloides) showed that 209 orthologous protein sequences of the oleaginous microbes were distributed over several processes of the cells. Based on the 41 sequences categorized by metabolism, putative routes potentially involved in the generation of precursors for fatty acid and lipid synthesis, particularly acetyl-CoA, were then identified that were not present in the non-oleaginous strains. We found a set of the orthologous oleaginous proteins that was responsible for the biosynthesis of this key two-carbon metabolite through citrate catabolism, fatty acid β-oxidation, leucine metabolism and lysine degradation. Our findings suggest a relationship between carbohydrate, lipid and amino acid metabolism in the biosynthesis of acetyl-CoA, which contributes to the lipid production of oleaginous microbes.
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Genome-wide phylogenetic analysis of differences in thermotolerance among closely related Acetobacter pasteurianus strains
Acetobacter pasteurianus is a Gram-negative strictly aerobic bacterium that is widely used for the industrial production of vinegar. Three Acetobacter pasteurianus strains, SKU1108, NBRC 3283 and IFO 3191, have the same 16S rRNA sequence (100 % sequence identity) but show differences in thermotolerance. To clarify the relationships between phylogeny and thermotolerance of these strains, genome-wide analysis of these three strains was performed. Concatenated phylogenetic analysis of a dataset of 1864 orthologues has shown that the more thermotolerant strains, SKU1108 and NBRC 3283, are more closely related to each other than to the more thermosensitive strain, IFO 3191. In addition, we defined a dataset of 2010 unique orthologues among these three strains, and compared the frequency of amino acid mutations among them. Genes involved in translation, transcription and signal transduction are highly conserved among each unique orthologous dataset. The results also showed that there are several genes with increased mutation rates in IFO 3191 compared with the thermotolerant strains, SKU1108 and NBRC 3283. Analysis of the mutational directions of these genes suggested that some of them might be correlated with the thermosensitivity of IFO 3191. Concatenated phylogenetic analysis of these closely related strains revealed that there is a phylogenetic relationship associated with this phenotype among the thermotolerant and thermosensitive strains.
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- Microbial Pathogenicity
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Blood–brain barrier invasion by Cryptococcus neoformans is enhanced by functional interactions with plasmin
More LessCryptococcus neoformans can invade the central nervous system through diverse mechanisms. We examined a possible role for host plasma proteases in the neurotropic behaviour of this blood-borne fungal pathogen. Plasminogen is a plasma-enriched zymogen that can passively coat the surface of blood-borne pathogens and, upon conversion to the serine protease plasmin, facilitate pathogen dissemination by degrading vascular barriers. In this study, plasminogen-to-plasmin conversion on killed and viable hypoencapsulated strains of C. neoformans required the addition of plasminogen activator (PA), but this conversion occurred in the absence of supplemented PA when viable strains were cultured with brain microvascular endothelial cells (BMEC). Plasmin-coated C. neoformans showed an enhanced invasive ability in Matrigel invasion assays that was significantly augmented in the presence of BMEC. The invasive effect of plasmin required viable pathogen and correlated with rapid declines in BMEC barrier function. Plasmin-enhanced invasion was inhibited by aprotinin, carboxypeptidase B, the lysine analogue epsilon-aminocaproic acid, and by capsule development. C. neoformans caused plasminogen-independent declines in BMEC barrier function that were associated with pathogen-induced host damage; however, such declines were significantly delayed and less extensive than those observed with plasmin-coated pathogen. BMEC adhesion and damage by hypoencapsulated C. neoformans were diminished by capsule induction but unaltered by plasminogen and/or PA. We conclude that hypoencapsulated C. neoformans can invade BMEC by a plasmin-dependent mechanism, in vitro, and that small, or minimal, surface capsule expression during the blood-borne phase of cryptococcosis may promote virulence by means of plasmin(ogen) acquisition.
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The two-component QseBC signalling system regulates in vitro and in vivo virulence of Aeromonas hydrophila
More LessWe recently demonstrated that the N-acyl-homoserine lactone [autoinducer (AI)-1] and LuxS (AI-2)-based quorum-sensing (QS) systems exerted positive and negative regulation, respectively, on the virulence of a diarrhoeal isolate SSU of Aeromonas hydrophila. However, the role of a newly identified, two-component-based QseBC QS system in the regulation of bacterial virulence in general is not well understood, with only a limited number of studies showing its function in bacterial pathogenesis. In this report, we identified and characterized the QseBC QS system in A. hydrophila SSU and found that, as was the case with enterohaemorrhagic Escherichia coli, the open reading frames for the qseB (the response regulator) and qseC (the sensor histidine kinase) genes overlapped by 4 bp at the ATGA motif. Our data provide evidence that deletion of the qseB gene from A. hydrophila resulted in attenuation of bacterial virulence in a septicaemic mouse model of infection and diminished swimming and swarming motility, and the mutant bacteria formed denser biofilms compared with those from the parental strain of A. hydrophila. The decrease in the virulence of the A. hydrophila ΔqseB mutant correlated with reduced production of protease and the cytotoxic enterotoxin, which has associated haemolytic activity. The swimming and swarming motility, haemolytic activity, protease production and biofilm formation were restored in the qseBC-complemented strain to a level similar to that of the wild-type A. hydrophila SSU. Our study is the first, to our knowledge, to report a functional QseBC QS system in A. hydrophila which may be linked to AI-1 and AI-2 QS systems in modulating bacterial virulence, possibly through the cyclic diguanosine monophosphate.
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- Physiology and Biochemistry
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l-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate
More LessCatabolism of amino acids is a central process in cellular nitrogen turnover, but only a few of the mechanisms involved have been described from basidiomycete fungi. This study identified one such mechanism, the l-amino acid oxidase (Lao1) enzyme of Hebeloma cylindrosporum, by 2D gel separation and MS. We determined genomic DNA sequences of lao1 and part of its upstream gene, a putative pyruvate decarboxylase (pdc2), and cloned the cDNA of lao1. The two genes were also identified and annotated from the genome of Laccaria bicolor. The lao1 and pdc2 gene structures were conserved between the two fungi. The intergenic region of L. bicolor possessed putative duplications not detected in H. cylindrosporum. Lao1 sequences possessed dinucleotide-binding motifs typical for flavoproteins. Lao1 was less than 23 % identical to Lao sequences described previously. Recombinant Lao1 of H. cylindrosporum was expressed in Escherichia coli, purified and refolded with SDS to gain catalytic activity. The enzyme possessed broad substrate specificity: 37 l-amino acids or derivatives served as effective substrates. The highest activities were recorded with l-glutamate, but positively charged and aromatic amino acids were also accepted. Michaelis constants for six amino acids varied from 0.5 to 6.7 mM. We have thus characterized a novel type of Lao-enzyme and its gene from the basidiomycete fungus H. cylindrosporum.
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Dynamics of a starvation-to-surfeit shift: a transcriptomic and modelling analysis of the bacterial response to zinc reveals transient behaviour of the Fur and SoxS regulators
More LessWe describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 µM Zn to Escherichia coli growing in severely Zn-depleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14- to 28-fold greater after Zn addition but there was also 6- to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.
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Effect of respiration and manganese on oxidative stress resistance of Lactobacillus plantarum WCFS1
More LessLactobacillus plantarum is a facultatively anaerobic bacterium that can perform respiration under aerobic conditions in the presence of haem, with vitamin K2 acting as a source of menaquinone. We investigated growth performance and oxidative stress resistance of Lb. plantarum WCFS1 cultures grown in de Man, Rogosa and Sharpe (MRS) medium without and with added manganese under fermentative, aerobic, aerobic with haem, and respiratory conditions. Previous studies showed that Lb. plantarum WCFS1 lacks a superoxide dismutase and requires high levels of manganese for optimum fermentative and aerobic growth. In this study, respiratory growth with added manganese resulted in significantly higher cell densities compared to the other growth conditions, while without manganese added, similar but lower cell densities were reached. Notably, cells derived from the respiratory cultures showed the highest hydrogen peroxide resistance in all conditions tested, although similar activity levels of haem-dependent catalase were detected in cells grown under aerobic conditions with haem. These results indicate that oxidative stress resistance of Lb. plantarum is affected by respiratory growth, growth phase, haem and manganese. As levels of haem and manganese can differ considerably in the raw materials used in fermentation processes, including those of milk, meat and vegetables, the insight gained here may provide tools to increase the performance and robustness of starter bacteria.
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