Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases

Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l-peptidyl donors, d-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d-form of incorporating amino acid acceptor occurs during or after peptide bond formation. l-peptidyl donors and d-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process.     

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [¹⁴C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed. Received: 9 July 1997 / Accepted: 3 November 1997     

Modulation of cell surface hydrophobicity in the benthic cyanobacterium Phormidium J-1

A shift from cell-surface hydrophobicity to hydrophilicity was experimentally induced in the benthic hydrophobic cyanobacterium Phormidium sp. strain J-1, by mechanical shearing, chloramphenicol, and proteolytic treatment after preincubation with sodium dodecyl sulfate (SDS). Treatment with SDS alone, while releasing large amounts of protein and carbohydrates from the cell wall, did not affect cell surface hydrophobicity.Ultrastructural analysis showed the cells, to be enveloped by a double-layered minicapsule. Treatments affecting cellsurface hydrophobicity also caused changes in capsular components. A model, describing cell-surface structure, composition and properties in Phormidium J-1, was constructed by correlating ultrastructural data with surface properties.Abbreviations SDS Sodium dodecyl sulfate - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea This paper is contributed in honor of Prof. G. Drews on the occasion of his sixtieth birthday     

Phase partition of gaseous hexane and surface hydrophobicity of Fusarium solani when grown in liquid and solid media with hexanol and hexane

The filamentous fungus, Fusarium solani, was grown in liquid and solid culture with glucose, glycerol, 1-hexanol and n-hexane. The partition coefficient with gaseous hexane (HPC) in the biomass was lower when grown in liquid medium with 1-hexanol (0.4) than with glycerol (0.8) or glucose (1) The HPC for surface growth were 0.2 for 1-hexanol, 0.5 for glycerol, 0.6 for glucose, and 0.2 for F. solani biomass obtained from a biofilter fed with gaseous n-hexane. These values show a 200-fold increase in n-hexane solubility when compared to water (HPC = 42). Lower HPC values can be partially explained by increased lipid accumulation with 1-hexanol, 10.5% (w/w) than with glycerol (8.5% w/w) or glucose (7.1% w/w). The diameter of the hyphae diminished from 3 μm to 2 μm when F. solani was grown on solid media with gaseous n-hexane thereby doubling the surface area for gaseous substrate exchange. The surface hydrophobicity of the mycelia increased consistently with more hydrophobic substrates and the contact angle of a drop of water on the mycelial mat was 113° when grown on n-hexane as compared to 75° with glucose. The fungus thus adapts to hydrophobic conditions and these changes may explain the higher uptake of gaseous hydrophobic substances by fungi in biofilters.     

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.     

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with ~ 45% of its normal content. ESI–MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or γ-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.     

Overexpression of wild-type PKD2 leads to increased proliferation and invasion of BON endocrine cells

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2WT) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.     

Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.     

通过组织培养筛选小麦抗赤霉病突变体的研究

选用中抗赤霉病的春小麦品种和品系的花药进行离体培养,以小麦赤霉病29号菌株产生的致病培养滤液为选择剂,结合物理诱变处理,进行抗病鉴定,用赤霉病菌分生孢子直接接种在愈伤组织和再生植株筛选抗病突变体。在83块愈伤中有53块抗病。在11株的再生植株中有9株均比未经培养滤液处理的对照提高了抗病性,从中选出4株抗病性接近或超过“苏麦3号”品种。     

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.     

Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities

The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1+ and TG2+. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 degrees C) and sub-optimal (18, 14 and 10 degrees C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 degrees C and 18 degrees C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants.     

Relocation of genes generates non-conserved chromosomal segments in Fusarium graminearum that show distinct and co-regulated gene expression patterns

Background

Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of Fusarium graminearum to elucidate the significance of non-conserved regions.

Results

The genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster.

Conclusions

Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-191) contains supplementary material, which is available to authorized users.     

Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation

β-Casein (β-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel β-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k_c, μM⁻¹), thermal aggregation constant (k_T, °C⁻¹) and aggregation rate constant (k_t, min⁻¹) were measured. Bovine β-CN displays greater chaperone activity than camel β-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine β-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel β-CN. Greater relative hydrophobicity of bovine β-CN than camel β-CN may be a factor responsible for stronger interactions of bovine β-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine β-CN.     

The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution

The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., H?lter, K., Correia, T., Arntz, C., Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133-141] was explored by chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence (extending over 68.5kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is determined by evolution of NRPS module specificity.