Conservation of griseofulvin genes in the gsf gene cluster among fungal genomes.

The polyketide griseofulvin is a natural antifungal compound and research in griseofulvin has been key in establishing our current understanding of polyketide biosynthesis. Nevertheless, the griseofulvin gsf biosynthetic gene cluster (BGC) remains poorly understood in most fungal species, including Penicillium griseofulvum where griseofulvin was first isolated. To elucidate essential genes involved in griseofulvin biosynthesis, we performed third-generation sequencing to obtain the genome of P. griseofulvum strain D-756. Furthermore, we gathered publicly available genome of 11 other fungal species in which gsf gene cluster was identified. In a comparative genome analysis, we annotated and compared the gsf BGC of all 12 fungal genomes. Our findings show no gene rearrangements at the gsf BGC. Furthermore, seven gsf genes are conserved by most genomes surveyed whereas the remaining six were poorly conserved. This study provides new insights into differences between gsf BGC and suggests that seven gsf genes are essential in griseofulvin production.

Purification and partial characterization of a thermostable antimicrobial protein from Bacillus subtilis FB123.

Antimicrobial proteins/peptides have attracted much attention because of their potential use in the industrial setting. In the present study, a thermostable antimicrobial protein (BSAMP) was purified from the culture supernatant of Bacillus subtilis FB123 by ammonium sulfate precipitation, gel chromatography on Sephacryl S-200 High Resolution, and ion exchange chromatography on DEAE Sepharose Fast Flow column. The molecular weight of the purified BSAMP was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the absence and presence of ß-mercaptoethanol. Its isoelectric point was determined to be 5.24 by isoelectric focusing electrophoresis. Periodic acid-Schiff staining revealed BSAMP to be a glycoprotein. Maximum activity was obtained at pH 6.0, with over 79% maximum activity retained at pH 3.0-5.0 and pH 7.0-9.0, respectively. BSAMP was shown to be highly thermostable, as its activity did not change obviously after treatment at 100 °C. However, it was partially sensitive to papain, trypsin, and alkali proteases. Finally, the bacterial protein exhibited broad-spectrum antimicrobial activity against several pathogenic organisms. These findings suggested that BSAMP should be further developed as a natural antibacterial agent for disease prevention in aquiculture and agriculture.

Control of grain size and rice yield by GL2-mediated brassinosteroid responses.

Given the continuously growing population and decreasing arable land, food shortage is becoming one of the most serious global problems in this century(1). Grain size is one of the determining factors for grain yield and thus is a prime target for genetic breeding(2,3). Although a number of quantitative trait loci (QTLs) associated with rice grain size have been identified in the past decade, mechanisms underlying their functions remain largely unknown(4,5). Here we show that a grain-length-associated QTL, GL2, has the potential to improve grain weight and grain yield up to 27.1% and 16.6%, respectively. We also show that GL2 is allelic to OsGRF4 and that it contains mutations in the miR396 targeting sequence. Because of the mutation, GL2 has a moderately increased expression level, which consequently activates brassinosteroid responses by upregulating a large number of brassinosteroid-induced genes to promote grain development. Furthermore, we found that GSK2, the central negative regulator of rice brassinosteroid signalling, directly interacts with OsGRF4 and inhibits its transcription activation activity to mediate the specific regulation of grain length by the hormone. Thus, this work demonstrates the feasibility of modulating specific brassinosteroid responses to improve plant productivity.

Cloning and expression of Aspergillus tamarii FS132 lipase gene in Pichia pastoris.

A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate.

[Rapid improvement of lipase production in Penicillium expansum by genome shuffling].

In the present study, the genome shuffling was used to improve lipase production of Penicillium expansum. A lipase producing mutant strain-Penicillium expansum FS8486 and a wild type of Aspergillus Tamarii FS-132 isolated from soil of a volcano in Xinjiang were used as the parental strains. After two rounds of genome shuffling, several elite daughter strains were screened. The lipase activity in one of the daughter strains was increased 317% over the starting strain FS8486. Comparisons of the morphology, RAPD (Random Amplification of Polymorphic DNA) polymorphism and the fatty acid compositions between the daughter and the parental strains suggested that the filial generation were generated by genome shuffling. In this study, the genome shuffling used successfully first time in eukaryotic microorganism and increases the production of the desired metabolite in short time, the study will be useful to spread the genome shuffling in eukaryotic microbial breeding.

Genetic variation in clones of Pseudomonas pseudoalcaligenes after ten months of selection in different thermal environments in the laboratory.

The random amplification of polymorphic DNA (RAPD) method was used to examine genetic variation in experimental clones of Pseudomonas pseudoalcaligenes in two experimental groups, as well as their common ancestor. Six clones derived from a single colony of P. pseudoalcaligenes were cultured in two different thermal regimes for 10 months. Three clones in the Control group were cultured at constant temperature of 35 degrees C and another three clones in the High Temperature (HT) group were propagated at incremental temperature ranging from 41 to 47 degrees C for 10 months. A total of 45 RAPD primers generated 146 polymorphic markers. Analysis of molecular variance (AMOVA) revealed mild (11%) but significant (P < 0.001) genetic difference between the Control and the HT clones. Phylogenetic analysis based on pairwise genetic distances showed that the HT clones were more divergent from the ancestor and from each other than the Control clones, implying that the HT clones of P. pseudoalcaligenes may have evolved faster than the Control clones.

Morphological changes of Pseudomonas pseudoalcaligenes in response to temperature selection.

Adaptation to novel environments usually entails morphological changes. The cell morphology of six experimental populations of Pseudomonas pseudoalcaligenes and their common ancestor were examined with scanning electron microscopy (SEM). The six experimental populations were propagated under different temperatures for 10 months: three of them cultured at constant normal temperature (35 degrees C) forming the control group, and the other three cultured at incremental higher temperatures (from 41 degrees to 47 degrees C) as the HT group. SEM showed the deformed and elongated cells in the 6-h cultures of both ancestral and control populations at 45 degrees C, indicating that 45 degrees C is stressful for the ancestral and the control populations. In contrast, the HT populations retained normal cell shape in the 6-h cultures at both 35 degrees C and 45 degrees C. The mean cell volumes of control and HT populations increased 29% and 34%, respectively, relative to the ancestor at their respective thermal regimens, suggestion that the culturing conditions might favor larger cells.

Changes in growth parameters of Pseudomonas pseudoalcaligenes after ten months culturing at increasing temperature.

Abstract In this paper, we report the thermal adaptation of Pseudomonas pseudoalcaligenes, characterized as changes in growth parameters. Six clones derived from a single colony of P. pseudoalcaligenes were cultured in two different temperature regimes for 10 months, with three clones forming the control group, cultured at a constant temperature, and another three clones forming the high-temperature (HT) group, cultured at increasing temperature (from 41 to 47 degrees C). Three growth parameters were measured: the lag time (lambda), which is the period between the time of transfer to a new medium and the time when the cell replication starts; the maximum growth rate (mu(m)); and the maximum yield (A). These three parameters are major components of bacterial fitness. The Gompertz and logistic models were used to estimate these three parameters. The two models gave almost identical estimates, but the Gompertz model had R(2) values consistently larger than the logistic model. The HT clones had significantly shorter lambda, but higher mu(m) and A than the control clones when both were grown at the originally stressful temperature of 45 degrees C, suggesting significant thermal adaptation. Interestingly, the HT clones grew equally well as the control clones at 35 degrees C, i.e. improved performance at 45 degrees C was not associated with a reduced performance at 35 degrees C.