Investigation of Arthrobacter spp. plasmids

During this work large molecular weight plasmids from Arthrobacter sp. 68b, A. rhombi PRH1 and VP3 bacteria were investigated as well as small plasmids from both A. rhombi strains. It was determined that genes encoding degradation proteins of phthalic acid, 2 methylpyridine and pyridine are located on plasmid p2MP (113 kb) in Arthrobacter sp. 68b. The degradation of phthalate, pyridine and its derivative is inducible process. It was proved that cells pre-grown with phthalic acid are able to utilise quinolinic acid. Monooxygenase, that cleaves pyridine ring between C2, C3 atoms, is induced by pyridine and 2 methylpyridine. The degradation pathway of mentioned compounds was proposed. Succinate semialdehyde and succinic acid are formed during utilisation. Genes, encoding 2-hydroxypyridine degradation proteins, are located on large molecular weight plasmid. The phenotype of large A. rhombi VP3 plasmid was not determined. Small plasmids from both A. rhombi strains were sequenced, open reading frames were determined and identified. Using the minimal replicon of small A. rhombi PRH1 plasmid, hybrid vectors pRMU824, pRMU824Km and pRMU824Tc were constructed for functional gene screening in Arthrobacter spp. and Rhodococcus spp. bacteria

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

A rapid method for the selection of amidohydrolases from metagenomic libraries by applying synthetic nucleosides and a uridine auxotrophic host

In this study, the development of a rapid, high-throughput method for the selection of amide-hydrolysing enzymes from the metagenome is described. This method is based on uridine auxotrophic Escherichia coli strain DH10B ∆pyrFEC and the use of N4-benzoyl-2’-deoxycytidine as a sole source of uridine in the minimal microbial M9 medium. The approach described here permits the selection of unique biocatalysts, e.g., a novel amidohydrolase from the activating signal cointegrator homology (ASCH) family and a polyethylene terephthalate hydrolase (PETase)-related enzyme

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N 4-acylcytosine derivatives

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (kcat/Km up to 2.8 × 106 M−1 s−1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases