Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence

Abstract

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the ␤-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the ␤-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent ␤-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…Evidence suggests that larger targeting cassettes facilitate the recombination in R. erythropolis AN12, as the integration event of the pJY39 rescue construct was readily generated (>500 Gm R CFU with 0.1 g DNA), whereas the traA gene disruption event was rare. Currently, the gene-disruption frequency of our method (1%) is poor compared to the 12% success rate reported by the -red-mediated gene disruption method (Patrauchan et al, 2005). In the future, we seek to optimize our procedure by exploring the eVects of increasing lengths of homology, the restriction site used, and by improving transformation eYciencies.…”

“…Four methods of targeted gene disruption that rely on double cross-over (ends-out) recombinations have been described for Rhodococcus genetic analysis prior to this work (Desomer et al, 1991;Patrauchan et al, 2005;Plaggenborg et al, 2006;van der Geize et al, 2001). One recent study of carotenoid production in R. erythropolis ATCC47072 successfully employed a simple single cross-over (ends-in) recombination strategy with a targeting cassette of 800 bp homologous sequence to generate a gene knockout (Tao and Cheng, 2004).…”

Detection of lung cancer using weighted digital analysis of breath biomarkers

“…Induced cytochrome p450 mixed oxidase activity could potentially modulate the catabolism of many of these breath VOCs, and thereby account for the large and diverse sets of candidate breath biomarkers associated with lung cancer. Cytochrome p450 enzymes catabolize most of the VOCs listed in Table 1, including isopropyl alcohol [17], hexanedione [18], camphor [19], benzophenone [20] and derivatives of tetroxane [21], benzene [22], benzoic acid [23], furan [24] and ionone [25] (this list is not exhaustive). The resulting diversity of candidate biomarkers constitutes a major strength of breath testing for lung cancer, since it ensures redundancy and robustness of the predictive algorithm.…”

Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…Data accumulated from basic studies on the characteristic features of rhodococci and the increasing knowledge obtained from the genome databases have enhanced the development of genetic manipulation techniques used for rhodococci analysis. Such techniques include the development of shuttle vectors using cryptic and/or antibiotic-resistant plasmids derived from Rhodococcus strains 10,19,20) , efficient transformation techniques using electroporation 21,22) , the expression vectors for protein production 23,24) , random mutagenesis methods using transposons or spontaneous illegitimate recombination 25,26) , and targeted gene disruption systems 11,27) . These techniques along with increasing genome information are enabling cell engineering in a manner such that there is an improvement in the useful properties of rhodococci such as their ability to act as biocatalysts; this improvement is due to the activation of catabolic pathways and/or depletion of the undesirable genes.…”