Construction of Escherichia coli-Arthrobacter-Rhodococcus shuttle vectors based on a cryptic plasmid from Arthrobacter rhombi and investigation of their application for functional screening

Abstract

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.

Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

“…The opening of the epoxide to a diol was presumed to be a plausible pathway for indole-2,3-dihydrodiol formation (44). Moreover, the oxidation of several N-heterocyclic compounds catalyzed by monooxygenases when two oxygen atoms (one from oxygen, another from water) were inserted was described recently (45)(46)(47). A similar mechanism for bacterial degradation of indole acetic acid was described (48) involving the intermediate dioxindole-3-acetic acid (DOAA), a structural analogue of 3-hydroxyindolin-2-one in indole degradation.…”

The use of pAL5000 Replicon Vectors to Transform Rhodococcus and GordoniaSpecies

“…Rhodococcus and Gordonia species face the same limitation although the number of vectors developed for these genera is on the increase (Dabbs et al, 1990;Bahn et al, 2005;Matsui et al, 2007;Stanislauskiene et al, 2012). However, Mycobacterium sp.…”

Bioinformatics-based molecular classification of Arthrobacter plasmids

“…Most of them have been constructed using the replication origin from cryptic plasmids of bacteria phylogenetically related to Arthrobacter species such as Corynebacterium [10,11] or Brevibacterium [10,12]. Although the vectors p54 [13] and pRH [2] have been isolated from Arthrobacter species, these are cryptic plasmids too, replicating also in Rhodococcus species. Most of the fully sequenced Arthrobacter strains harbor 1 to 3 plasmids of varying size from 2 kb to more than 300 kb.…”

“…Representatives of this genus are often found in both contaminated and pristine soils, where they represent an important fraction of the culturable bacteria [2]. Some notable features of arthrobacters are their changing shape during the life cycle from rod shaped to coccoid bacteria, their resistance to desiccation [3], ionizing radiation [4] and cold [5] as well as their ability to metabolize a wide range of natural and man-made compounds, including nicotine, phenanthrene [6], 4-chlorophenol [7], dimethyl sulfone [8] and atrazine [9].…”