Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

Degradation of monoaromatics by Bacillus pumilus MVSV3

ABSTRACT Monoaromatics, such as benzene, toluene, ethylbenzene and xylene (BTEX), are simple aromatic compound that are highly toxic due to their high solubility nature. Many chemical and physical methods for their degradation and breakup into nontoxic products are available, but still use of microorganism is preferred over these processes. In this present study Bacillus pumilus MVSV3 (Accession number JN089707), a less explored bacteria in the field of BTEX degradation, isolated from petroleum contaminated soil is utilized for BTEX degradation. At optimized conditions the isolate degraded 150 mg/L of BTEX completely within 48 h. GC-MS analysis revealed that the microorganism produces catechol and muconic acid during degradation indicating an ortho pathway of degradation. Enzyme assays were carried out to identify and characterize catechol 1, 2- dioxygenase (C12D). The optimal temperature and pH for the enzyme activity was identified as 35 (C and 7.5, respectively. SDS-PAGE revealed the molecular weight of the enzyme to be approximately 35,000 Da. Zymography analysis indicated the presence of three isoforms of the enzyme. Hence Bacillus pumilus MVSV3 and the isolated C12D, proved to be efficient in degrading the toxic aromatic compounds.

A novel benzoate-degrading Rhodococcus strain contains three catA genes with one being transcriptionally active during the growth on benzoate.

The gene encoding catechol 1, 2-dioxygenase (catA) is one of the common biomarkers used for evaluation of microbial degradation of aromatic compounds and bioremediation potentials, whereas the expression of the gene has been little studied. The diversity of the catA genes and their expression pattern in a novel Rhodococcus sp. strain LIN isolated from coastal sediments of the Bohai Sea, China, were investigated. We found that this strain exhibited catechol 1, 2-dioxygenase activity when cultivated with benzoate as the sole carbon source. The catA genes were amplified with degenerate primers and sequenced. Sequence analyses show that the strain has three catA genes which are highly similar to each other (> 97%). However, only one gene was transcribed according to reverse transcription-polymerase chain reaction (RT-PCR) analysis. This indicates that RNA-based approaches could better reflect the diversity and scale of the environmental microbial function than DNA-based ones, when sequencing and quantifying (e.g. quantitative PCR) of the catA gene are employed to assess the environmental pollutions and bioremediations.