Sequence characterization and computational analysis of the non-ribosomal peptide synthetases controlling biosynthesis of lipopeptides, fengycins and bacillomycin D, from Bacillus amyloliquefaciens Q-426

Lipopeptides secreted by bacteria attract interest because of their uses in biomedicine, biotechnology and food technology; however, harnessing their megasynthases (non-ribosomal peptide synthetases, NRPSs) has met with some difficulties in heterologous expression and crystallization. Here, we used similarity and phylogenetic analysis of NRPS sequences, including the fengycin and iturin family synthetases from Bacillus spp., and have developed a novel approach for delineating the length and boundaries of NRPS domains from Bacillus amyloliquefaciens strain Q-426. The sequences were further characterized (including specific residues and conserved motifs) that gave insight into the basis of the substrate specificity. Data from the prediction of the NRPS domains, obtained by the self-optimized prediction method with Alignment program, showed they are all structurally unstable, making it difficult to determine their crystal structures.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

EP2 Receptor Signaling Pathways Regulate Classical Activation of Microglia

Activation of EP2 receptors by prostaglandin E2 (PGE₂) promotes brain inflammation in neurodegenerative diseases, but the pathways responsible are unclear. EP2 receptors couple to Gα_s and increase cAMP, which associates with protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factors (Epacs). Here, we studied EP2 function and its signaling pathways in rat microglia in their resting state or undergoing classical activation in vitro following treatment with low concentrations of lipopolysaccharide and interferon-γ. Real time PCR showed that PGE₂ had no effect on expression of CXCL10, TGF-β1, and IL-11 and exacerbated the rapid up-regulation of mRNAs encoding cyclooxygenase-2, inducible NOS, IL-6, and IL-1β but blunted the production of mRNAs encoding TNF-α, IL-10, CCL3, and CCL4. These effects were mimicked fully by the EP2 agonist butaprost but only weakly by the EP1/EP3 agonist 17-phenyl trinor PGE₂ or the EP4 agonist CAY10598 and not at all by the EP3/EP1 agonist sulprostone and confirmed by protein measurements of cyclooxygenase-2, IL-6, IL-10, and TNF-α. In resting microglia, butaprost induced cAMP formation and altered the mRNA expression of inflammatory mediators, but protein expression was unchanged. The PKA inhibitor H89 had little or no effect on inflammatory mediators modulated by EP2, whereas the Epac activator 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate acetoxymethyl ester mimicked all butaprost effects. These results indicate that EP2 activation plays a complex immune regulatory role during classical activation of microglia and that Epac pathways are prominent in this role.     

Phenazine-1-carboxylic acid production in a chromosomally non-scar triple-deleted mutant Pseudomonas aeruginosa using statistical experimental designs to optimize yield

We constructed a non-scar triple-deleted mutant Pseudomonas aeruginosa to improve phenazine-1-carboxylic acid (PCA) yield and then optimized the culture conditions for PCA production. Using a non-scar deletion strategy, the 5′-untranslated region of the phz1 gene cluster and two genes, phzM and phzS, were knocked out of the P. aeruginosa strain M18 genome. The potential ability for high-yield PCA production in this triple-deleted mutant M18MSU1 was successfully realized by using statistical experimental designs. A 2^5–1 fractional factorial design was used to show that the three culture components of soybean meal, corn steep liquor and ethanol had the most significant effect on PCA production. Using a central composite design, the concentration of the three components was optimized. The maximum PCA production was predicted to be 4,725.1 mg/L. With the optimal medium containing soybean meal 74.25 g/L, corn steep liquor 13.01 g/L and ethanol 21.84 ml/L, a PCA production of 4,771.2 mg/L was obtained in the validation experiments, which was nearly twofold of that before optimization and tenfold of that in the wild-type strain. This non-scar triple-deleted mutant M18MSU1 may be a suitable strain for industrial production of this biologically synthesized fungicide due to its high PCA production, presumed safety, thermal adaptability and cost-effectiveness.     

Overwintering strategy of endoparasitoids in Chilo suppressalis: a perspective from the cold hardiness of a host

Although parasitoids ultimately kill their host, koinobiont parasitoids must protect not only themselves but also their hosts against extreme environments. In this study, the parasitism rate of Chilo suppressalis Walker (Lepidoptera: Pyralidae) was investigated, and the average body weights, supercooling points, and concentrations of glycerol (acting as a cryoprotectant) in the hemolymph were compared between parasitized and non‐parasitized larvae. Five species of koinobiont endoparasitoids parasitized the overwintering C. suppressalis larvae and the total parasitism rate was 47.6% (n = 1 537). Average body weight of parasitized larvae was significantly lower than that of non‐parasitized larvae, and the parasitism rate of the lighter group (20–30 mg) was highest. The supercooling point of parasitized C. suppressalis larvae (?15.7 ± 0.3 °C) was significantly lower than that of the non‐parasitized larvae (?14.3 ± 0.2 °C). In addition, supercooling points were not correlated with body weights between parasitized and non‐parasitized larvae, indicating that cold hardiness of parasitized larvae was enhanced by endoparasitoids. Furthermore, the concentration of glycerol in the hemolymph was significantly higher in parasitized larvae (205.0 ± 7.1 μmol ml^?1) than in non‐parasitized larvae (169.8 ± 14.4 μmol ml^?1), which suggests that the mechanism that decreases the supercooling point of parasitized larvae was associated with glycerol. All these results indicated that the cold hardiness of parasitized C. suppressalis larvae was enhanced by their endoparasitoids, which benefitted overwintering endoparasitoids.     

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.     

Impact of GC content on gene expression pattern in chicken

Background

GC content varies greatly between different genomic regions in many eukaryotes. In order to determine whether this organization named isochore organization influences gene expression patterns, the relationship between GC content and gene expression has been investigated in man and mouse. However, to date, this question is still a matter for debate. Among the avian species, chicken (Gallus gallus) is the best studied representative with a complete genome sequence. The distinctive features and organization of its sequence make it a good model to explore important issues in genome structure and evolution.

Methods

Only nuclear genes with complete information on protein-coding sequence with no evidence of multiple-splicing forms were included in this study. Chicken protein coding sequences, complete mRNA sequences (or full length cDNA sequences), and 5^′ untranslated region sequences (5^′ UTR) were downloaded from Ensembl and chicken expression data originated from a previous work. Three indices i.e. expression level, expression breadth and maximum expression level were used to measure the expression pattern of a given gene. CpG islands were identified using hgTables of the UCSC Genome Browser. Correlation analysis between variables was performed by SAS Proprietary Software Release 8.1.

Results

In chicken, the GC content of 5^′ UTR is significantly and positively correlated with expression level, expression breadth, and maximum expression level, whereas that of coding sequences and introns and at the third coding position are negatively correlated with expression level and expression breadth, and not correlated with maximum expression level. These significant trends are independent of recombination rate, chromosome size and gene density. Furthermore, multiple linear regression analysis indicated that GC content in genes could explain approximately 10% of the variation in gene expression.

Conclusions

GC content is significantly associated with gene expression pattern and could be one of the important regulation factors in the chicken genome.