Study on the osmoregulation of "Halomonas socia" NY-011 and the degradation of organic pollutants in the saline environment.

"Halomonas socia" NY-011, a new species of moderately halophilic bacteria isolated and identified in our laboratory, can grow in high concentrations of salt ranging from 0.5 to 25%. In this study, the whole genome of NY-011 was sequenced and a detailed analysis of the genomic features was provided. Especially, a series of genes related to salt tolerance and involved in xenobiotics biodegradation were annotated by COG, GO and KEGG analyses. Subsequently, RNA-Seq-based transcriptome analysis was applied to explore the osmotic regulation of NY-011 subjected to high salt stress for different times (0 h, 1 h, 3 h, 6 h, 11 h, 15 h). And we found that the genes related to osmoregulation including excluding Na+ and accumulating K+ as well as the synthesis of compatible solutes (alanine, glutamate, ectoine, hydroxyectoine and glycine betaine) were up-regulated, while the genes involved in the degradation of organic compounds were basically down-regulated during the whole process. Specifically, the expression trend of genes related to osmoregulation increased firstly then dropped, which was almost opposite to that of degrading organic pollutants genes. With the prolongation of osmotic up-shock, NY-011 survived and gradually adapted to osmotic stress, the above-mentioned two classes of genes slowly returned to normal expression level. Then, the scanning electron microscope (SEM) and transmission electron microscope (TEM) were also utilized to observe morphological properties of NY-011 under hypersaline stress, and our findings showed that the cell length of NY-011 became longer under osmotic stress, at the same time, polyhydroxyalkanoates (PHAs) were synthesized in the cells. Besides, physiological experiments confirmed that NY-011 could degrade organic compounds in a high salt environment. These data not only provide valuable insights into the mechanism of osmotic regulation of NY-011; but also make it possible for NY-011 to be exploited for biotechnological applications such as degrading organic pollutants in a hypersaline environment.

Discovery and characterizaton of a novel lipase with transesterification activity from hot spring metagenomic library.

A new gene encoding a lipase (designated as Lip-1) was identified from a metagenomic bacterial artificial chromosome(BAC) library prepared from a concentrated water sample collected from a hot spring field in Niujie, Eryuan of Yunnan province in China. The open reading frame of this gene encoded 622 amino acid residues. It was cloned, fused with the oleosin gene and over expressed in Escherichia coli to prepare immobilized lipase artificial oil body AOB-sole-lip-1. The monomeric Sole-lip-1 fusion protein presented a molecular mass of 102.4 kDa. Enzyme assays using olive oil and methanol as the substrates in petroleum ether confirmed its transesterification activity. Hexadecanoic acid methyl ester, 8,11-Octadecadienoic acid methyl ester, 8-Octadecenoic acid methyl ester, and Octadecanoic acid methyl ester were detected. It showed favorable transesterification activity with optimal temperature 45 °C. Besides, the maximal biodiesel yield was obtained when the petroleum ether system as the organic solvent and the substrate methanol in 350 mmol/L (at a molar ratio of methanol of 10.5:1) and the water content was 1%. In light of these advantages, this lipase presents a promising resource for biodiesel production.

Antifungal Activity of Eucalyptus Oil against Rice Blast Fungi and the Possible Mechanism of Gene Expression Pattern.

Eucalyptus oil possesses a wide spectrum of biological activity, including anti-microbial, fungicidal, herbicidal, acaricidal and nematicidal properties. We studied anti-fungal activities of the leaf oil extracted from Eucalyptus. grandis × E. urophylla. Eleven plant pathogenic fungi were tested based on the mycelium growth rates with negative control. The results showed that Eucalyptus oil has broad-spectrum inhibitory effects toward these fungi. Remarkable morphological and structural alterations of hypha have been observed for Magnaporthe grisea after the treatment. The mRNA genome array of M. grisea was used to detect genes that were differentially expressed in the test strains treated by the Eucalyptus oil than the normal strains. The results showed 1919 genes were significantly affected, among which 1109 were down-regulated and 810 were up-regulated (p < 0.05, absolute fold change >2). According to gene ontology annotation analysis, these differentially expressed genes may cause abnormal structures and physiological function disorders, which may reduce the fungus growth. These results show the oil has potential for use in the biological control of plant disease as a green biopesticide.

[Homologous cloning and expression of PDS gene from Dunaliella salina in Escherichia coli].

OBJECTIVE: Phytoene desaturase PDS is a eukaryotic nuclear membrane binding protein, we used different expression methods to search for the soluble expression strategy of membrane binding protein in Escherichia coli. METHODS: We cloned the full-length cDNA sequence of PDS from Dunaliella salina through RACE. First, we utilized prokaryotic expression vector pET-28a to construct pET-28a-PDS vector. Then, we substituted PLtac promoter for T7 promoter in pET-28a to construct pET-PLtac-PDS vector. Last, we constructed pET-Mistic-PDS fusion vector by integrating Mistic sequence into pET-28a. All were transformed into BL21(DE3) for protein expression. RESULTS: The 2237-bp full-length cDNA sequence of PDS was cloned, including a 1749-bp open reading frame, encoding 582 amino acids (NCBI accession: GQ923693.1). The expression of PDS protein was low via pET-28a-PDS and pET-PLtac-PDS vector, and proteins were mostly expressed in inclusion body. The expression of PDS protein was significantly increased via pET-Mistic-PDS vector, in addition most were expressed as soluble protein which possessed dehydrogenase activity. CONCLUSION: Mistic as the solubilization label was able to promote proper folding of membrane proteins and improve solubility. Protease activity assay proved that Mistic could maintain the enzyme activity.

Halomonas socia sp. nov., isolated from high salt culture of Dunaliella salina.

A moderately halophilic bacteria designed strain NY-011(T) was isolated from the high salt culture of Dunaliella salina in Chengdu of Sichuan Province, China. The isolate was Gram-negative, nonmotile, rod-shaped and 12.5-21.6 µm in length. Colonies on solid media are circular, wet, smooth and cream. The strain grew optimally at 37 °C, pH 7.0 and in the presence of 8 % NaCl. Acid was produced from glycerol, D-arabinose, glucose, trehalose, inositol, mannose, mannitol, sucrose, maltose and sorbitol. Catalase is produced but not oxidase. The major fatty acids are C18: 1ω7c (37.59 %), C19: 0 cyclo ω8c (18.29 %), C16: 0 (16.05 %) and C6: 0 (12.43 %). The predominant respiratory lipoquinone found in strain NY-011(T) is ubiquinone with nine isoprene units (Q-9). The genomic DNA G + C content of strain NY-011(T) was 62.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NY-011(T) belonged to the genus Halomonas. The highest levels of 16S rRNA gene sequence similarity were found between the strain NY-011(T) and H. pantelleriensis (sequence similarity 98.43 %). However, the levels of DNA-DNA relatedness between them were only 23.1 %. In addition, the strain NY-011(T) had a phenotypic profile that readily distinguished it from H. pantelleriensis. The strain NY-011(T) therefore represents a new species of the genus Halomonas, for which the name Halomonas socia sp. nov. is proposed, with NY-011(T) (=CCTCC AB 2011033(T) = KCTC 23671(T)) as the type strain.

Cloning and characterization of a phosphate transporter gene in Dunaliella salina.

The full-length cDNA of a Na(+) -dependent Pi transport gene (DsSPT1) in Dunaliella salina was cloned by 3' and 5' Rapid Amplification of cDNA Ends (RACE), with an open reading frame (ORF) encoding 716 predicted amino acids, which exhibited 60.5% identity to that of Na(+) -dependent Pi transport 1 (DvSPT1) from Dunaliella viridis. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in DvSPT1 from D. viridis and PHO89 from Saccharomyces cerevisiae. The result of real-time quantitative PCR showed that expression level of DsSPT1 was enhanced at first and reached its peak at 90 min after salt stress; however, D. salina cells rapidly absorbed extracellular inorganic phosphorus which was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) during the first 5 min under salt stress. It suggested that D. salina on the absorption of inorganic phosphorus was regulated at DsSPTI posttranslational level.

Studies on a chlorogenic acid-producing endophytic fungi isolated from Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a traditional medicinal plant of China, and it is one of the main sources of chlorogenic acid. Chlorogenic acid is an ester of caffeic acid, quinic acid, and a phenolic compound that has antibacterial, antifungal, antioxidant, and antitumor activities. The purpose of this study was to determine whether endophytic fungi isolated from Eucommia ulmoides Oliver had the same ability to produce chlorogenic acid. Primary screening was done by antibacterial and antifungal reactions, and the strain reselection was done with high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. Extracts of the leaf and cortex of Eucommia ulmoides Oliver were also deteted by HPLC, then positive results of HPLC were analyzed by GC-MS and LC-MS. In this study, 29 strains were isolated from Eucommia ulmoides Oliver. Most of them had antibacterial activity, and a few of them had antifungal activity. One ingredient of the B5 extract had a retention time identical to that of authentic chlorogenic acid. With GC-MS, other ingredients, isocoumarin and p-chlorocinnamide, were found. With LC-MS, chlorogenic acid and geniposide related to Eucommia ulmoides Oliver were found. The strain B5 was identified as Sordariomycete sp. Thus, endophytic fungi may produce the bioactive compound chlorogenic acid, as their host plant does, and could be used for the production of chlorogenic acid by fermentation in the future.

Identification of a gene encoding the light-harvesting chlorophyll a/b proteins of photosystem I in green alga Dunaliella salina.

There are four LhcII genes of Dunaliella salina have been submitted to the database of GenBank. However, little is known about Lhca genes of this green alga, although this knowledge might be available to study the composition and phylogenesis of Lhc gene family. Recently, one Lhca gene was been cloned from the green alga D. salina by PCR amplification using degenerate primers. This cDNA, designated as DsLhca1, contains an open reading frame encoded a protein of 222 amino acids with a calculated molecular mass of 27.8 kDa. DsLhca1 is predicted to contain three transmembrane domains and a N-terminal chloroplast transit peptide (cTP) with length of 33 amino acids. The genomic sequence of DsLhca1 is composed of five introns. The deduced polypeptide sequence of this gene showed a lower degree of identity (less than 30%) with LHCII proteins from D. salina. But its homology to Lhca proteins of other algae (Volvox carteri Lhca_AF110786) was higher with pairwise identities of up to 67.1%. Phylogenetic analysis indicated that DsLhcal protein cannot be assigned to any types of Lhca proteins in higher plants or in Chlamydomonas reinhardtii.

Isolation of a FAD-GPDH gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase from Dunaliella salina.

The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH), recently reported in plants, has been detailed in yeast and animal systems. It oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate (DHAP) on the outer surface of mitochondrial inner membrane. A cDNA encoding the Dunaliella salina mitochondrial glycerol-3-phosphate dehydrogenase (DsFAD-GPDH) has been cloned and sequenced. The full length cDNA is 2791 bp, with an open reading frame (ORF) encoding 650 predicted amino acids, which show strong homology to reported FAD-GPDHs and have an apparent mitochondrial targeting sequence in the N-terminal. The sequence has been submitted to the GenBank database under Accession No. DQ916107. Results of Real-Time Quantitative PCR and enzymatic assays show that expression of DsFAD-GPDH is enhanced at first by salt treatment, and repressed by oxygen deficiency and cold stress.

Cloning, expression, and functional characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichia coli.

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.

Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina.

A cDNA encoding a nicotinamide adenine dinucleotide (NAD+) -dependent glycerol 3-phosphate dehydrogenase (GPDH) has been cloned by rapid amplification of cDNA ends from Dunaliella salina. The cDNA is 3032 base pairs long with an open reading frame encoding a polypeptide of 701 amino acids. The polypeptide shows high homology with published NAD+ -dependent GPDHs and has at its N-terminal a chloroplast targeting sequence. RNA gel blot analysis was performed to study GPDH gene expression under different conditions, and changes of the glycerol content were monitored. The results indicate that the cDNA may encode an osmoregulated isoform primarily involved in glycerol synthesis. The 701-amino-acid polypeptide is about 300 amino acids longer than previously reported plant NAD+ -dependent GPDHs. This 300-amino-acid fragment has a phosphoserine phosphatase domain. We suggest that the phosphoserine phosphatase domain functions as glycerol 3-phosphatase and that, consequently, NAD+ -dependent GPDH from D. salina can catalyze the step from dihydroxyacetone phosphate to glycerol directly. This is unique and a possible explanation for the fast glycerol synthesis found in D. salina.

Identification of two genes encoding the major light-harvesting chlorophyll a/b proteins of photosystem II in green alga Dunaliella salina.

Dunaliella salina is a useful model for studying the respective roles of each LHCII protein at the molecular level in extreme environmental conditions. However, information about the LhcII genes in D. salina is very limited. In order to identify more LhcII genes in D. salina, two additional LhcII cDNAs were obtained by screening a cDNA library. The genomic DNA was amplified by PCR using a specific primer set corresponding to the 5' and 3' untranslated regions of each transcript. The untranslated regions of the two additional genes are obviously different from each other; therefore they are two genes. Each gene contains an open reading frame for a protein of 253 amino acids. The two deduced proteins in D. salina are 99% identical at the amino acid sequence level to the previously reported LHCII protein in the same genus D. tertiolecta. Unrooted phylogenetic tree showed that types of LHCII proteins in D. salina did not correspond to any types in C. reinhardtii.