Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis.

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD's SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD's SP were also higher than the other two strains containing PhoC or ZMO0331's SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.

Exploring the polyadenylated RNA virome of sweet potato through high-throughput sequencing.

BACKGROUND: Viral diseases are the second most significant biotic stress for sweet potato, with yield losses reaching 20% to 40%. Over 30 viruses have been reported to infect sweet potato around the world, and 11 of these have been detected in China. Most of these viruses were detected by traditional detection approaches that show disadvantages in detection throughput. Next-generation sequencing technology provides a novel, high sensitive method for virus detection and diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: We report the polyadenylated RNA virome of three sweet potato cultivars using a high throughput RNA sequencing approach. Transcripts of 15 different viruses were detected, 11 of which were detected in cultivar Xushu18, whilst 11 and 4 viruses were detected in Guangshu 87 and Jingshu 6, respectively. Four were detected in sweet potato for the first time, and 4 were found for the first time in China. The most prevalent virus was SPFMV, which constituted 88% of the total viral sequence reads. Virus transcripts with extremely low expression levels were also detected, such as transcripts of SPLCV, CMV and CymMV. Digital gene expression (DGE) and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that the highest viral transcript expression levels were found in fibrous and tuberous roots, which suggest that these tissues should be optimum samples for virus detection. CONCLUSIONS/SIGNIFICANCE: A total of 15 viruses were presumed to present in three sweet potato cultivars growing in China. This is the first insight into the sweet potato polyadenylated RNA virome. These results can serve as a basis for further work to investigate whether some of the 'new' viruses infecting sweet potato are pathogenic.

Scanning of transposable elements and analyzing expression of transposase genes of sweet potato [Ipomoea batatas].

BACKGROUND: Transposable elements (TEs) are the most abundant genomic components in eukaryotes and affect the genome by their replications and movements to generate genetic plasticity. Sweet potato performs asexual reproduction generally and the TEs may be an important genetic factor for genome reorganization. Complete identification of TEs is essential for the study of genome evolution. However, the TEs of sweet potato are still poorly understood because of its complex hexaploid genome and difficulty in genome sequencing. The recent availability of the sweet potato transcriptome databases provides an opportunity for discovering and characterizing the expressed TEs. METHODOLOGY/PRINCIPAL FINDINGS: We first established the integrated-transcriptome database by de novo assembling four published sweet potato transcriptome databases from three cultivars in China. Using sequence-similarity search and analysis, a total of 1,405 TEs including 883 retrotransposons and 522 DNA transposons were predicted and categorized. Depending on mapping sets of RNA-Seq raw short reads to the predicted TEs, we compared the quantities, classifications and expression activities of TEs inter- and intra-cultivars. Moreover, the differential expressions of TEs in seven tissues of Xushu 18 cultivar were analyzed by using Illumina digital gene expression (DGE) tag profiling. It was found that 417 TEs were expressed in one or more tissues and 107 in all seven tissues. Furthermore, the copy number of 11 transposase genes was determined to be 1-3 copies in the genome of sweet potato by Real-time PCR-based absolute quantification. CONCLUSIONS/SIGNIFICANCE: Our result provides a new method for TE searching on species with transcriptome sequences while lacking genome information. The searching, identification and expression analysis of TEs will provide useful TE information in sweet potato, which are valuable for the further studies of TE-mediated gene mutation and optimization in asexual reproduction. It contributes to elucidating the roles of TEs in genome evolution.

Transcriptome analysis to identify putative floral-specific genes and flowering regulatory-related genes of sweet potato.

Sweet potato flowers were collected for a transcriptome analysis to identify the putative floral-specific and flowering regulatory-related genes by using the RNA-sequencing technique. Pair-end short reads were de novo assembled by an integrated strategy, and then the floral transcriptome was carefully compared with several published vegetative transcriptomes. A total of 2595 putative floral-specific and 2928 putative vegetative-specific transcripts were detected. We also identified a large number of transcripts similar to the key genes in the flowering regulation network of Arabidopsis thaliana.

Draft genome sequence of Enterobacter cloacae subsp. cloacae strain 08XA1, a fecal bacterium of giant pandas.

Enterobacter cloacae, a common pathogenic bacterium, is a Gram-negative bacillus. We analyzed the draft genome of Enterobacter cloacae subsp. cloacae strain 08XA1 from the feces of a giant panda in China. Genes encoding a ß-lactamase and efflux pumps, as well as other factors, have been found in the genome.

Draft genome sequence of Bacillus pumilus BA06, a producer of alkaline serine protease with leather-dehairing function.

Bacillus pumilus BA06 was isolated from the proteinaceous soil and produced an extracellular alkaline protease with leather-dehairing function. The genome of BA06 was sequenced. The comparative genome analysis indicated that strain BA06 is different in genome from the other B. pumilus strains, with limited insertions, deletions, and rearrangements.

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy.

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl(2) to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl(2) induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl(2)-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl(2)-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.

Digital gene expression analysis based on integrated de novo transcriptome assembly of sweet potato [Ipomoea batatas (L.) Lam].

BACKGROUND: Sweet potato (Ipomoea batatas L. [Lam.]) ranks among the top six most important food crops in the world. It is widely grown throughout the world with high and stable yield, strong adaptability, rich nutrient content, and multiple uses. However, little is known about the molecular biology of this important non-model organism due to lack of genomic resources. Hence, studies based on high-throughput sequencing technologies are needed to get a comprehensive and integrated genomic resource and better understanding of gene expression patterns in different tissues and at various developmental stages. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end (PE) RNA-Sequencing was performed, and generated 48.7 million of 75 bp PE reads. These reads were de novo assembled into 128,052 transcripts (≥ 100 bp), which correspond to 41.1 million base pairs, by using a combined assembly strategy. Transcripts were annotated by Blast2GO and 51,763 transcripts got BLASTX hits, in which 39,677 transcripts have GO terms and 14,117 have ECs that are associated with 147 KEGG pathways. Furthermore, transcriptome differences of seven tissues were analyzed by using Illumina digital gene expression (DGE) tag profiling and numerous differentially and specifically expressed transcripts were identified. Moreover, the expression characteristics of genes involved in viral genomes, starch metabolism and potential stress tolerance and insect resistance were also identified. CONCLUSIONS/SIGNIFICANCE: The combined de novo transcriptome assembly strategy can be applied to other organisms whose reference genomes are not available. The data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest in sweet potato. Characterization of sweet potato transcriptome provides an effective tool for better understanding the molecular mechanisms of cellular processes including development of leaves and storage roots, tissue-specific gene expression, potential biotic and abiotic stress response in sweet potato.

Modulated expression of genes associated with NO signal transduction contributes to the cholesterol-lowering effect of electro-acupuncture.

Electro-acupuncture (EA) at Fenglong acupoint (ST40) can lower the levels of serum cholesterol and triacylglycerols. To study the hepatic genes responsible for the cholesterol-lowering effect of EA, suppression subtractive hybridization combined with the switch mechanism at the 5'-end of RNA template cDNA synthesis and long-distance PCR were employed using hepatic tissues from hypercholesterolemia and EA-treated mice. 68 % of the identified genes are involved in metabolism, immune response, and signal transduction pathways. Real-time PCR and western blot indicate that EA at ST40 induces the expression of nNOS and Mt1, two genes involved in NO signal transduction. EA treatment for hypercholesterolemia thus involves the modulation of several biological pathways and provides a physiological link between NO signal transduction and the cholesterol-lowering effect of EA.

[Cloning and optimizing expression of a periplasmic solute-binding gene gsiB from Escherichia coli].

Cloning and expression of gsiB was carried out for studying protein structure and function of glutathione transport system. The coding sequence of Escherichia coli gsiB that encodes the periplasmic solute-binding protein of a glutathione transporter was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring GFP reporter gene through the method Sequence and Ligation-Independent Cloning (SLIC). The resulting recombinant plasmid pWaldo-GFP-GsiB was transformed into different E. coli strains and the expression conditions were optimized. It was found that E. coli BL21(DE3) was the best strain for gsiB gene expression among the four strains tested. Induction at lower incubation temperature of 18 degrees C and 0.1 mmol/L of IPTG led to higher expression of gsiB in E. coli BL21(DE3). Western blotting analysis also showed the expression of gsiB and the molecular weight of expressed protein as expected.

Gene expression in secondary metabolism and metabolic switching phase of Phanerochaete chrysosporium.

Ligninolytic enzymes are well-known to play the crucial roles in lignin biodegradation and have potential applications in industrial processes. The filamentous white-rot fungus, Phanerochaete chrysosporium, has been widely used as a model organism for studying these ligninolytic enzymes that are able to degrade the lignin during the secondary metabolism. To study the gene expression in secondary metabolism and metabolic switching phase of P. chrysosporium, we constructed a metabolic-switching phase suppression subtractive hybridization (SSH) cDNA library and a secondary metabolic phase SSH cDNA library to compare their mRNA expression profiles. We isolated the genes that are specially expressed and subsequently identified four genes that specially expressed during metabolic-switching phase while 22 genes in secondary metabolic phase. Accordingly, these specially expressed genes might play key roles in different metabolic stages, which would offer more new insights into the shift from nitrogen to lignin metabolism.

Characterization of a unique proline iminopeptidase from white-rot basidiomycetes Phanerochaete chrysosporium.

A putative gene encoding proline iminopeptidase (PchPiPA) was cloned from Phanerochaete chrysosporium BKM-F-1767 by RT-PCR and expressed successfully in Escherichia coli. The cDNA is 942 bp in length and encodes 313 amino acids. The recombinant enzyme was only able to hydrolyze Pro-pNA among the tested synthetic substrates. There is no activity detected toward Leu-pNA, Phe-pNA and Tyr-pNA, as well as GGG-pNA, SGR-pNA, AAV-pNA, AAPL-pNA, AAVA-pNA. And the recombinant enzyme could cleave the peptides derived from enzyme-hydrolytic natural proteins to release free lysine, which was confirmed using synthetic oligopeptides with lysine at N termini as substrate. The optimal pH and temperature for this enzyme were 8.0 and 45 degrees C, respectively. The catalytic activity was inhibited slightly by Mg(2+), Al(3+), Ca(2+), Fe(3+), Fe(2+) and Ba(2+); strongly by Ni(2+), Mn(2+) and Co(2+), and almost inactivated by Zn(2+), Cu(2+) and Hg(2+). In addition, the enzyme was not sensitive to EDTA-Na(2), as well as redoxes of DTT, beta-ME and H(2)O(2). The protease inhibitors of benzamidine hydrochloride and phenylmethyl sulfonyfluoride caused a moderate inhibition. The V(max), K(m) and k(cat) toward Pro-pNA were 347.86 mumol min(-1) mg(-1), 2.15 mM and 218.10 S(-1), respectively. The deduced catalytic triad of Ser(107), Asp(264) and His(292) was confirmed by site-directed mutagenesis because the individual replacement of Ser(107) to Asp, Asp(264) to Ala or His(292) to Leu led complete inactivation. Transcriptional analysis by RT-PCR showed that PchPiPA could be expressed under ligninolytic and non-ligninolytic conditions. Conclusively, it was suggested that the proline iminopeptidase may be a member of the proteolytic system in this fungus. The availability of recombinant protein may be potentially used in certain proteolytic processing.

Tea catechins induce the conversion of preformed lysozyme amyloid fibrils to amorphous aggregates.

Natural polyphenols are major constituents of plant foods and herbs. Numerous studies have demonstrated that natural polyphenols inhibited amyloid formation and destabilized the preformed amyloid fibrils. However, the molecular mechanism for the antiamyloidogenesis of polyphenols is still unclear and remains to be further explored. In the present study, the preformed lysozyme fibrils were used as an in vitro model to study the disruptive effects of tea catechins on amyloid fibrils. Results showed that tea catechins induced the conversion of lysozyme fibrils to amorphous aggregates and inhibited fibril-induced hemolysis. Hydroquinone also showed disruptive effect on the fibrils, whereas phenol and two typical antioxidants, ascorbic acid and alpha-tocopherol, did not affect the fibrillar structure, suggesting that polyphenolic structure is essential for fibril deposition. Correlation analyses indicate that the fibril-depositing effects were related to both the antioxidative potency and hydrophobicity of tea catechins. These findings provide new evidence for comprehensive understanding of the interaction between natural polyphenols and amyloid fibrils.

[Stress-responsive expression analysis of glutathione-S-transferase gene of Ipomoea batatas (L.) Lam].

A prokaryotic expression plasmid pET-IbGST, which contains the full encoding region of a glutathione-S- transferase (GST) gene of sweet potato, was constructed. The recombinant IBGSTU1 protein was expressed in Escherichia coli and found in the soluble fraction, as well as in insoluble inclusion bodies of lysed cells. Its enzymatic activity was detected using UV spectrophotometer. The protein was purified and used to prepare antibody. Semi-quantitative RT-PCR and Western blotting analyses demonstrated that IBGSTU1 gene was not expressed under normal conditions. When subjected to some environ-mental stresses such as cold-stress or heavy-medal stress, the organism switches on the expression of IBGSTU1 at both mRNA and protein levels, and its expression has tissue specificity.

[Molecular cloning and expression of alcohol dehydrogenase gene of Phanerochaete chrysosporium].

When Phanerochaete chrysosporium is grown under oxygen-limited condition, ethanol is one of the important metabolites. In order to understand the metabolic mechanism of P. chrysosporium grown under oxygen-limited condition, a cDNA sequence (1 071 bp) designated "PCAdh1" encoding an alcohol dehydrogenase (ADH) was cloned from the filamentous white-rot fungus P. chrysosporium. PCAdh1 gene encodes a protein of 356 amino acid residues. Although the catalytic domain and coenzyme-binding domain were highly conserved, the protein sequence of PCAdh1 showed a low level of similarity to other known ADH. The recombinant PCAdh1 protein was expressed in Escherichia coli and its enzyme activity was detected. The protein was purified and used to prepare antibody. Semi-quantitative RT-PCR and Western blot demonstrated that the expression level of PCAdh1 in P. chrysosporium remained stable despite the lowered oxygen content, indicating that the gene expression is constitutive. But with the reduction of oxygen content, the overall activity of ADH from the crude mycelia proteins was increased during the growing periods, implying that the expression of other Adh genes in P. chrysosporium is inductive.

Substrate specificity and thermostability of the dehairing alkaline protease from Bacillus pumilus.

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k(m) value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 degrees C, and lost activity so quickly above 70 degrees C. The thermostability was largely dependent on metal ion, especially Ca(2+), although other ions, like Mg(2+), Mn(2+), and Co(2+), could sustain stability at certain extent within limited time. Cu(2+), Fe(2+), as well as Al(3+), did not support the enzyme to retain activity at 60 degrees C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.

Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli.

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.

[Site-directed mutagenesis and function analysis of glgC gene from Escherichia coli.].

Using genomic DNA of Escherichia coli JM109 as a template, glgC gene was amplified by polymerase chain reaction (PCR). The full coding sequence of this gene is 1296 bp. To get 3 mutants that amino acids changed: P295S (V121A, M151I, V334D), G336D and P295S/G336D (K109R) by recombinant PCR, respectively named 295+3, 336 and 295/336+1. The 3 mutants and the original glgC were subcloned into the prokaryotic expression vector pET-32a, and these recombinant expression plasmids were transformed into E. coli BL21 (DE3) for effective expression. The host cells were induced with IPTG and then identified by SDS-PAGE. A specific fused-expression product 67 kDa was detected, which was the same as the deduced protein. In the host cells above, the biological activities of the expressed products were detected by iodine vapor staining and glycogen content testing. The host cell transformed with the mutated gene-336 had higher glycogen content, which was identical to the gene-295/336+1. This confirmed that Pro295Ser could not reinforce the decrease of the feedback inhibition effect of the AGPase. Meanwhile, another host cell transformed with the mutated gene-295+3 showed decreased glycogen rather than the expected increasing glycogen. This might be caused by another mutation, Val334Asp in gene-295+3, which might induce the change of the allosteric region of the objective protein.

Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif.

A novel bacterial cell-surface display system was developed in Escherichia coli using omp1, a hypothetical outer membrane protein of Zymomonas mobilis. By using this system, we successfully expressed beta-amylase gene of sweet potato in E. coli. The display of enzyme on the membrane surface was also confirmed. The recombinant beta-amylase showed to significantly increase hydrolytic activity toward soluble starch. Our results provide a basis for constructing an engineered Z. mobilis strain directly fermenting raw starch to produce ethanol.