Tetramethylpyrazine-Inducible Promoter Region from Rhodococcus jostii TMP1.

An inducible promoter region, PTTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of PTTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of PTTMP were determined using site-directed mutagenesis. Furthermore, PTTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.

Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.

Identification and characterization of a tetramethylpyrazine catabolic pathway in Rhodococcus jostii TMP1.

At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.

Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.

Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.

Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries.

A high-throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester-hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2',3',5'-O-tri-acetyluridine and 2',3',5'-O-tri-hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.

Ketoreductase TpdE from Rhodococcus jostii TMP1: characterization and application in the synthesis of chiral alcohols.

Background. Production of highly pure enantiomers of vicinal diols is desirable, but difficult to achieve. Enantiomerically pure diols and acyloins are valuable bulk chemicals, promising synthones and potential building blocks for chiral polymers. Enzymatic reduction of ketones is a useful technique for the synthesis of the desired enantiomeric alcohols. Here, we report on the characterization of a ketoreductase TpdE from Rhodococcus jostii TMP1 that is a prospective tool for the synthesis of such compounds. Results. In this study, NADPH-dependent short-chain dehydrogenase/reductase TpdE from Rhodococcus jostii TMP1 was characterized. The enzyme exhibited broad substrate specificity towards aliphatic 2,3-diketones, butan-3-one-2-yl alkanoates, as well as acetoin and its acylated derivatives. TpdE stereospecifically reduced α-diketones to the corresponding diols. The GC-MS analysis of the reduction products of 2,3- and 3,4-diketones indicated that TpdE is capable of reducing both keto groups in its substrate leading to the formation of two new chiral atoms in the product molecule. Bioconversions of diketones to corresponding diols occurred using either purified enzyme or a whole-cell Escherichia coli BL21 (DE3) biocatalyst harbouring recombinant TpdE. The optimum temperature and pH were determined to be 30-35 °C and 7.5, respectively. Conclusions. The broad substrate specificity and stereoselectivity of TpdE from Rhodococcus jostii TMP1 make it a promising biocatalyst for the production of enantiomerically pure diols that are difficult to obtain by chemical routes.