Expression and subcellular localization of organophosphate hydrolase in acephate-degrading Pseudomonas sp. strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides.

UNLABELLED: Organophosphate hydrolase (OPH), the product of an organophosphate-degrading (opd) gene cloned from Brevundimonas diminuta, hydrolyses the triester linkage found in neurotoxic organophosphate (OP) insecticides and nerve agents. Despite the fact that OPHs have a broad substrate range, OP compounds with a P-S linkage, such as insecticides like acephate, are poor substrates for the enzyme. Expression of OPH in acephate-utilizing Pseudomonas sp. Ind01 generated a live biocatalyst capable of degrading a wide range of OP insecticides. The heterologously expressed OPH, which is a substrate of twin arginine transport (Tat) pathway, successfully targeted to the membrane of Pseudomonas sp. Ind01. The membrane-associated OPH had a size that coincided with the mature form of OPH (mOPH), suggesting successful processing and targeting of the expressed OPH to the membrane. Pseudomonas sp. Ind01 expressing OPH degraded a variety of OP insecticides besides using acephate as sole carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalyst capable of degrading a wide range of organophosphate (OP) insecticides was generated by expressing an organophosphate degradation gene in Pseudomonas sp. Ind01 involved in mineralization of acephate. The biocatalyst can be used to eliminate a wide range of OP insecticide residues from the environment.

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

AIM: To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. METHODS AND RESULTS: A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. CONCLUSIONS: Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Generation of continuous packed bed reactor with PVA-alginate blend immobilized Ochrobactrum sp. DGVK1 cells for effective removal of N,N-dimethylformamide from industrial effluents.

Effective removal of dimethylformamide (DMF), the organic solvent found in industrial effluents of textile and pharma industries, was demonstrated by using free and immobilized cells of Ochrobactrum sp. DGVK1, a soil isolate capable of utilizing DMF as a sole source of carbon, nitrogen. The free cells have efficiently removed DMF from culture media and effluents, only when DMF concentration was less than 1% (v/v). Entrapment of cells either in alginate or in polyvinyl alcohol (PVA) failed to increase tolerance limits. However, the cells of Ochrobactrum sp. DGVK1 entrapped in PVA-alginate mixed matrix tolerated higher concentration of DMF (2.5%, v/v) and effectively removed DMF from industrial effluents. As determined through batch fermentation, these immobilized cells have retained viability and degradability for more than 20 cycles. A continuous packed bed reactor, generated by using PVA-alginate beads, efficiently removed DMF from industrial effluents, even in the presence of certain organic solvents frequently found in effluents along with DMF.

Complete mineralisation of dimethylformamide by Ochrobactrum sp. DGVK1 isolated from the soil samples collected from the coalmine leftovers.

A bacterial strain DGVK1 capable of using N,N-dimethylformamide (DMF) as sole source of carbon and nitrogen was isolated from the soil samples collected from the coalmine leftovers. The molecular phylogram generated using the complete sequence of 16S rDNA of the strain DGVK1 showed close links to the bacteria grouped under Brucellaceae family that belongs to alphaproteobacteria class. Specifically, the 16S rDNA sequence of strain DGVK1 has shown 97% similarity to Ochrobactrum anthropi LMG 3331 (D12794). This bacterium has also shown impressive growth on dimethylamine, methylamine, formaldehyde and formate that are considered to be the prominent catabolic intermediates of DMF. DMF degradation has led to the accumulation of ammonia and dimethylamine contributing to the increase of pH of the medium. The DMF-grown resting cells of Ochrobactrum sp. DGVK1 have also contributed for the release of ammonia when resting cell suspension was added to phosphate buffer containing DMF. Similar experiments done with the glucose-grown cultures have not produced ammonia and thus indicating the inducible nature of DMF-degrading enzymes in Ochrobactrum sp. DGVK1. Further, dimethylformamidase, dimethylamine dehydrogenase and methylamine dehydrogenase, the key enzymes involved in the degradation of DMF, were assayed, and the activities of these enzymes were found only in DMF-grown cultures further confirming the inducible nature of the DMF degradation. Based on these results, DMF degradation pathway found in Ochrobactrum sp. DGVK1 has been proposed.

Plasmid mediated organophosphate pesticide degradation by Flavobacterium balustinum.

A bacterium capable of degrading methyl parathion, an organophosphorus insecticide into paranitrophenol (as evidenced by TLC) and other metabolites, was isolated from the agricultural soils of Anantapur district, Andhra Pradesh, India. This bacterium, identified as Flavobacterium balustinum was found to harbour an indigenous plasmid of approximately 86 kb in size. The degradative enzyme, parathion hydrolase, was found to be encoded by this plasmid. No enzyme activity was observed in plasmid cured strain.

Structure of the nifQ gene from Enterobacter agglomerans 333 and its overexpression in Escherichia coli.

The nifQ gene, involved in early stages of iron-molybdenum cofactor (FeMo-co) biosynthesis, was identified downstream of the nifB and nifF genes of Enterobacter agglomerans. This gene was cloned and its nucleotide sequence determined. The amino acid sequence, as deduced from the nucleotide sequence, revealed an accumulation of cysteine amino acid residues at the C-terminal end of the protein. The cysteine cluster showed the following consensus sequence Cys-X4-Cys-X2-Cys-X5-Cys, which is a typical characteristic of metal-binding proteins. Further, the nifQ gene was cloned downstream of strong transcriptional (bacteriophage lambda PLPR) and translational (atpE) signals of the expression vector pCYTEXP1 and expressed as an unfused, soluble protein in Escherichia coli. The molecular mass of 19 kDa, as deduced by SDS-PAGE, is in good agreement with the molecular mass deduced from the nucleotide sequence. The availability of high-level expression clones should facilitate purification of large quantities of the recombinant NifQ protein and elucidation of its properties.

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription.

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3'-region of the nifM gene, the nifL and nifA genes and the 5'-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical sigma 54-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(x3) A(x3) G(x5)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH4+. Maximal promoter activity occurred at 25 degrees C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH4+ concentration in the medium exceeded 4 mM.

Self-transmissible nif plasmid (pEA9) of Enterobacter agglomerans 339: molecular cloning and evidence for the existence of similar nif clusters on dissimilar plasmids in Enterobacter strains.

A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.

Site-specific mutagenesis in Enterobacter agglomerans: construction of nif B mutants and analysis of the gene's structure and function.

A novel technique was developed which may be generally well suited to the site-specific construction of mutations in Enterobacter agglomerans. The method is based on the observation that E. agglomerans can be cured of a plasmid of the incompatibility group IncQ by cultivation on citrate-containing medium. To test the applicability of this technique, we inserted a kanamycin cassette into the cloned nifB gene, transferred it into E. agglomerans, and selected for recombinants in which the wild-type nifB was replaced by the mutated gene by growing transformants on citrate medium with kanamycin. The nifB- mutants with the kanamycin cassette inserted in either orientation showed a nif- phenotype. Further, we determined the nucleotide sequence of nifB. A typical sigma 54-dependent promoter and a consensus NifA binding site were found upstream of nifB. Activation of this promoter by both heterologous and homologous NifA proteins was observed in vivo. The predicted amino acid sequence of the NifB protein showed strong similarity to the NifB sequences of other diazotrophic bacteria. The typical clustering of cysteine residues at the N-terminal end indicates its involvement in Fe-Mo cofactor biosynthesis.