Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Post-synthetic acetylation of HMGB1 protein modulates its interactions with supercoiled DNA

High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1 affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating its interaction with negatively supercoiled DNA.     

Stability and structural constraints of random brain networks with excitatory and inhibitory neural populations

The stability of brain networks with randomly connected excitatory and inhibitory neural populations is investigated using a simplified physiological model of brain electrical activity. Neural populations are randomly assigned to be excitatory or inhibitory and the stability of a brain network is determined by the spectrum of the network’s matrix of connection strengths. The probability that a network is stable is determined from its spectral density which is numerically determined and is approximated by a spectral distribution recently derived by Rajan and Abbott. The probability that a brain network is stable is maximum when the total connection strength into a population is approximately zero and is shown to depend on the arrangement of the excitatory and inhibitory connections and the parameters of the network. The maximum excitatory and inhibitory input into a structure allowed by stability occurs when the net input equals zero and, in contrast to networks with randomly distributed excitatory and inhibitory connections, substantially increases as the number of connections increases. Networks with the largest excitatory and inhibitory input allowed by stability have multiple marginally stable modes, are highly responsive and adaptable to external stimuli, have the same total input into each structure with minimal variance in the excitatory and inhibitory connection strengths, and have a wide range of flexible, adaptable, and complex behavior.     

Influence of growth conditions on the nisin production of bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production of three bioengineered strains, (LAC338, LAC339 and LAC340) with immunity (nisFEG) and/or regulation (nisRK) genes of nisin biosynthesis on plasmids in the Lactococcus lactis LL27 nisin producer, was evaluated under pH-controlled and pH-uncontrolled batch fermentations. Optimization studies showed that fructose and yeast extract yielded the highest nisin activity. The strains LAC338, LAC339, and LAC340 produced 24, 45, and 44% more nisin, respectively, than wild-type L. lactis LL27 after 12-h incubation. However, sharp decreases in the yield of nisin were observed at the late phase of fermentation with LAC339 and LL27 in contrast to LAC340 and LAC338 strains for which the high level of nisin could be maintained longer. Obviously, increasing the copy number of the regulation genes together with immunity genes in the nisin producers retarded the loss of nisin in the late phase of the fermentation.     

Geographic variation in the growth of white croaker,Pennahia argentata,off the coast of northwest Kyushu,Japan

We examined geographic variation in the growth of white croaker,Pennahia argentata, off the coast of northwest Kyushu, Japan, Ariake Sound, Tachibana Bay, Omura Bay and the Goto Sea by examination of otoliths. The outer margins of the otoliths showed that a opaque zone was formed once a year, with its peak in June, and could be used as an annulus. White croaker caught during this study reached a maximum age of 10years in the Goto Sea. The growth curves for both sexes in all localities were expressed by the von Bertalanffy growth equations from back-calculated total length of fish. We found significant sexual differences in growth curves in Ariake Sound, Tachibana Bay and the Goto Sea. For both sexes, white croaker in the Goto Sea reached the largest length at each estimated age of the four localities. The growth curves for both sexes showed significant differences among four localities, suggesting that several stocks may exist in the study area although the greatest distance between each locality was at most 30 km.     

Quantitative determination of curcuminoids in Curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis

The three major curcuminoids, curcumin, demethoxycurcumin and bis-demethoxycurcumin, from Curcuma domestica Val. (Curcuma longa L.) and Curcuma xanthorrhiza Roxb. (Zingiberaceae) were fully separated and quantified in less than 5 min using a capillary zone electrophoresis method with standard fused-silica capillaries and photodiode array detection. An electrolyte solution of 20 mM phosphate, 50 mM sodium hydroxide and 14 mM beta-cyclodextrin was found to be appropriate. Quantification was performed with 3,4-dimethoxy-trans-cinnamic acid as internal standard, and the limit of detection was 0.01 mg/mL. Extraction, stabilisation during sample storage and quantification procedures were optimised and carried out with drugs and commercial curry powder from different provenances. The results were compared with the photometric method of the monograph Curcumae xanthorrhizae rhizoma of the European Pharmacopoeia.     

Structural changes in the digestive glands of larval Antarctic krill (<Emphasis Type="Italic">Euphausia superba</Emphasis>) during starvation

The effects of starvation on ultrastructure of digestive gland cells were studied in furcilia larvae of Antarctic krill (Euphausia superba: hereafter krill). Under laboratory conditions, larvae were starved for 0, 5, 10, 15, 20 and 25 days, and their R-cells were investigated by transmission electron microscope. R-cells are thought to play a role in the storage and absorption of nutrients. In fed larvae, numerous mitochondria scattered homogenously, and densely packed microvilli were observed on the apical surface of R-cells. After 5 days of starvation, mitochondria were swollen and were found concentrated in the apical region in R-cells. A decrease in cell volume and an increase in thickness of the basal lamina with many irregular infoldings were observed after 10–15 days of starvation. Lipid droplets were rarely found in the R-cells regardless of whether larvae had been fed or starved suggesting an inability to store lipid. Without the ability to store energy in the form of lipid, survival would be dependant on sourcing continuous food until maturation.     

Conserved motifs in mechanosensitive channels MscL and MscS

Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.