Nitrogen Metabolism of an Anoxygenic Filamentous Phototrophic Bacterium Oscillocholris trichoides Strain DG-6

Abstract— The possible nitrogen sources for Osc. trichoides DG6, a typical strain of the Oscillochloridaceae family, are ammonium, N2, glutamate, asparagine, glycine, and glutamine. The assimilation of molecular nitrogen occurs with the participation of nitrogenase, the structural gene of which, nifH, is located in the gene cluster which also includes the genes of the nifD and nifK nitrogenase subunits and the auxiliary nifB gene. Considering that nifHBDK clusters have been also annotated in the genomes of other members of the Oscillochloridaceae family, including uncultured and candidate taxa, it can be assumed that the ability to fix nitrogen is a property immanent for this entire family. The pathways for assimilating ammonium in the cells grown using different nitrogen sources may differ. Osc. trichoides DG6 growing in a medium containing ammonium assimilated it with the participation of glutamate dehydrogenase, which is determined by a single gene. The expression product of this gene has dual functionality and can be used to implement the reaction with both NAD and NADP. With the growth of Osc. trichoides DG6 on a medium with glutamate as the only nitrogen source all the enzymes necessary for the implementation of the GS‑GOGAT pathway were found in the cells. However, for the glutamine synthetase reaction, ammonium, which was absent in the growth medium, was required. The source of ammonium may be glutamate metabolized through glutamate dehydrogenase.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta)

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

A Novel MicroRNA From the Translated Region of the Giardiavirus rdrp Gene Governs Virus Copy Number in Giardia duodenalis

Giardia duodenalis is an important zoonotic parasite that can cause human and animal diarrhea. Giardiavirus (GLV) is a double-stranded RNA virus in Totiviridae family, which specifically infects trophozoites of the primitive protozoan parasite G. duodenalis. However, the GLV infectious and the pathogenicity of the G. duodenalis still remain to be confirmed. The GLV genome is 6,277 bp, which encodes two proteins (Gag and Gag-Pol). The expression of Gag-Pol protein is regulated by a-1 ribosomal frameshift. In this report, we identified a novel microRNA (GLV miRNA1) from the GLV. Split ligation northern results showed that GLV miRNA1 is a special expression product of GLV, and the precursor was also identified by primer extension. Antisense sequence of the GLV miRNA1 could increase the copy number of virus in G. duodenalis. It suggests that GLV miRNA1 governs the copy number of Giardiavirus in G. duodenalis. Most importantly, the GLV miRNA1 lies at the translated region of the rdrp gene, which is the first case that microRNA locates in the translated region of a known protein. It may be implying a novel phenomenon for miRNA biogenesis.

Introduction And Modern Historical Outline

The expression ‘product liability’ is usually understood to refer to the civil liability of manufacturers and others where damage or loss is caused by products which fail to meet the standards claimed expressly or impliedly for them or which are dangerous or otherwise defective. Such liability may arise either in contract or in tort and both aspects are covered in some detail in the course of this work. A further aspect which is discussed in less detail involves the use of the criminal law. Relevant provisions may apply either to particular types of product or be of more general application. The former are referred to only in passing whilst examples of the latter include Pt II of the Consumer Protection Act 1987 and other measures concerned with general product safety.

Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

To clone and express theγ-polyglutamic acid (γ-PGA) synthetase genepgsBCA inBacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA intoBacillus subtilisWB600.PgsBCAwas expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesizeγ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher.γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates ofγ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of theγ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing theγ-PGA synthetase genepgsBCAinB. subtilissystem has potential industrial applications.

Prokaryotic Expression and Identification on the ag85a and mpb70 Fusion Gene of Mycobacterium bovis

Based on splicing by overlapping extension (SOE) polymerase chain reaction (PCR) ,theag85aandmpb70were amplified and the fusion gene ag85a-mpb70 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-85a-70. pMD-85a-70 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified ag85a-mpb70 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-85a-70 was constructed. Plasmid containing pET-85a-70 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 49 kDa fusion protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that Ag85A-MPB70 was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of novel vaccine against bovine tuberculosis.

Fusion, Expression and Identification on the mpb51 and mpb63 Genes of Mycobacterium bovis

The DNA fragments of mpb51 and mpb63 were fused by splicing by overlapping extension(SOE) polymerase chain reaction(PCR),and the fusion gene mpb51-63 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-51-63. pMD-51-63 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified mpb51-63 fusion gene was subcloned into the expression vector pET28a(+),and the prokaryotic expression vector pET-51-63 was constructed. Plasmid containing pET-51-63 was transformed into competence Escherichia coli BL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 43ku exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity of Mycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of subunit vaccine, DNA vaccine and diagnostic reagents against bovine tuberculosis.