DNA methylation and mRNA expression of COL6A3 in antler mesenchyme of female and male reindeer.

BACKGROUD: Reindeer is the only deer species that both male and female produce antlers, which provides a particularly interesting case in studying the differences between antlers of the two sexes. Alpha 3(VI) Collagen Gene (COL6A3), forms a microfibrillar network associated with the structural integrity and biomechanical properties, has been found to be one of the differentially expressed genes in antler mesenchyme of female and male reindeer. OBJECTIVE AND METHODS: The promoter sequence of reindeer COL6A3 gene was obtained using the cloning technology and analyzed by the bioinformatics methods. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the COL6A3 promoter in reindeer antler mesenchyme. Real-time quantitative PCR was used to detect COL6A3 expression in the antler mesenchyme of female and male reindeer. RESULTS: Sequence analysis revealed that the reindeer COL6A3 partial promoter sequence was 983 bp including the possible promoter region at + 105 bp to + 155 bp. Homology and phylogenetic analysis indicated that the COL6A3 promoter of reindeer had the closest genetic distance with Bos taurus, Capra hircus and Ovis aries. BSP results indicated that the methylation level of COL6A3 promoter in the female reindeer antler mesenchyme was significantly higher than in the male. Correlating with increased methylation status, we also found that COL6A3 mRNA expression in female reindeer antler mesenchyme was significantly lower than in the male. CONCLUSION: The higher methylation level of the COL6A3 gene in female reindeer antler mesenchyme coincides with decreased COL6A3 mRNA expression, thereby affecting the transposon silencing mechanism and possibly contributing to apparent differences of antlers in female and male reindeer.

Analysis on Genetic Diversity of Reindeer (Rangifer tarandus) in the Greater Khingan Mountains Using Microsatellite Markers.

Jian-Cheng Zhai, Wei-Shi Liu, Ya-Jie Yin, Yan-Ling Xia, and He-Ping Li (2017) The only population of reindeer (Rangifer tarandus) in China, herded extensively by the Ewenki people, is the most southern population in the world. Genetic diversity plays a key role in the survival of endangered reindeer. To systematically understand the genetic variability of reindeer in China, 163 individuals from 8 populations were analyzed using 11 microsatellite loci. A total of 85 alleles were detected and the average number of alleles per locus was 7.7. The observed heterozygosity and expected heterozygosity ranged from 0.3736 to 0.5299 and from 0.6491 to 0.7608. Hardy-Weinberg equilibrium analysis indicated that a de ciency of heterozygotes existed in all eight populations. Both the FST and AMOVA analyses showed a low level of genetic di erentiation among populations. UPGMA dendrogram revealed that population SYL formed one cluster, separating from the other populations. Then the GWQ and YSH populations formed another cluster and clustered with the BDX, BLJY, DML, DW and MLYS populations. Increasing the current exchange rate of reindeer among different populations and establishing natural reserve may be the e ective approaches to conserve the fragile reindeer populations in China.

Production and characterization of high efficiency bioflocculant isolated from Klebsiella sp. ZZ-3.

In this study, a new bioflocculant (ZZ-3) is isolated and evaluated. This novel flocculant was derived Klebsiella, which was identified by 16S rDNA sequencing as well as biochemical and physiological analyses. The composition of ZZ-3 was found to be 84.6% polysaccharides and 6.1% protein. More specifically, the amount (moles) of the polysaccharides rhamnose, mannose, and galactose were found to be 6.48, 2.47, and 1.74 greater than glucose, respectively. Results show ZZ-3 has a relatively high molecular weight (603-1820 kDa) and contains many functional groups (hydroxyl, amide, carboxyl, and methoxyl) that likely contribute to flocculation. The results of microscopic observation, zeta potential measurements, and ZZ-3 bioflocculant structure suggested that bridging was the main mechanism for flocculation with kaolin. Based on a high flocculation efficiency, thermal stability, pH tolerance and the ability to flocculate without additional cations, ZZ-3 shows potential for industrial application.

[Advances in molecular mechanism of bacterial reduction of hexavalent chromium].

Cr(VI) has been causing serious environmental pollution due to its carcinogenicity, teratogenicity and strong migration. Reduction of Cr( VI) to Cr(III), a precipitation that is much less toxic, is an efficient strategy to control Cr pollution. Within the strategy, bacterial reduction of Cr(VI) to Cr(III) has been considered as one of the best bioremediation methods because of its efficiency, environment friendly, and low cost; however, the molecular mechanism remains large unknown. This review summarizes Cr(VI) reduction bacterial species and its application in pollution control, elaborates the pathways of Cr( VI) reduction and functional proteins involved, concludes the molecular mechanism of baterial reduction Cr(VI), and discusses the orientation of the future research.

Physiological roles of mycothiol in detoxification and tolerance to multiple poisonous chemicals in Corynebacterium glutamicum.

Mycothiol (MSH) plays important roles in maintaining cytosolic redox homeostasis and in adapting to reactive oxygen species in the high-(G + C)-content Gram-positive Actinobacteria. However, its physiological roles are ill defined compared to glutathione, the functional analog of MSH in Gram-negative bacteria and most eukaryotes. In this research, we explored the impact of intracellular MSH on cellular physiology by using MSH-deficient mutants in the model organism Corynebacterium glutamicum. We found that intracellular MSH contributes significantly to resistance to alkylating agents, glyphosate, ethanol, antibiotics, heavy metals and aromatic compounds. In addition, intracellular MSH is beneficial for withstanding oxidative stress induced by various oxidants in C. glutamicum. This study greatly expanded our current knowledge on the physiological functions of mycothiol in C. glutamicum and could be applied to improve the robustness of this scientifically and commercially important species in the future.

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038.

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.

Identification and quantification of mycothiol in Actinobacteria by a novel enzymatic method.

Mycothiol (MSH) was reported to be the dominant low molecular weight thiol in members of the Actinobacteria. In this study, a simple, fast, and sensitive method for qualitative and quantitative determination of MSH molecules was developed based on maleylpyruvate isomerase (MPI) from Corynebacterium glutamicum. The principle of this method is that the activity of MPI from C. glutamicum was dependent on MSH molecules. It was found that this MPI activity displayed a linear response (R (2) = 0.9928) at MSH amounts ranging from 0.12 to 3.98 pmol in the defined assay system. This observation was applied to calculate the MSH levels, and the newly developed method was compared with thiol-specific fluorescent-labeling high-performance liquid chromatography method. Forty-eight genera of Actinobacteria were screened for MSH and 43 genera were reported for MSH occurrence, and the MSH levels in Actinobacteria were determined to be 0.01 to 9.69 µmol/g of residual dry cell weight.

Crystal structures and site-directed mutagenesis of a mycothiol-dependent enzyme reveal a novel folding and molecular basis for mycothiol-mediated maleylpyruvate isomerization.

Mycothiol (MSH) is the major low molecular mass thiols in many Gram-positive bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. The physiological roles of MSH are believed to be equivalent to those of GSH in Gram-negative bacteria, but current knowledge of MSH is limited to detoxification of alkalating chemicals and protection from host cell defense/killing systems. Recently, an MSH-dependent maleylpyruvate isomerase (MDMPI) was discovered from C. glutamicum, and this isomerase represents one example of many putative MSH-dependent enzymes that take MSH as cofactor. In this report, fourteen mutants of MDMPI were generated. The wild type and mutant (H52A) MDMPIs were crystallized and their structures were solved at 1.75 and 2.05 A resolution, respectively. The crystal structures reveal that this enzyme contains a divalent metal-binding domain and a C-terminal domain possessing a novel folding pattern (alphabetaalphabetabetaalpha fold). The divalent metal-binding site is composed of residues His52, Glu144, and His148 and is located at the bottom of a surface pocket. Combining the structural and site-directed mutagenesis studies, it is proposed that this surface pocket including the metal ion and MSH moiety formed the putative catalytic center.

The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum.

Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC- and mshD- lost the ability to produce mycothiol, but mutant mshB- produced mycothiol as the wild type did. The phenotypes of mutants mshC- and mshD- were the same as the wild type when grown in LB or BHIS media, but mutants mshC- and mshD- were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 +/- 11.9 microM and 1520 +/- 57.4 micromol/min/mg, respectively (mycothiol concentration, 2.5 microM). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.