Dayananda Siddavattam

Biodegradation of methyl parathion and p-nitrophenol : evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (opd) Gene Cluster Found in Flavobacterium sp.

ABSTRACT Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved. Two opd plasmids, pPDL2 from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase. Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF. We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion. The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.

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.

Genome of a Novel Isolate of Paracoccus denitrificans Capable of Degrading N,N-Dimethylformamide

The bacterial genus Paracoccus is comprised of metabolically versatile organisms having diverse degradative capabilities and potential industrial and environmental applications for bioremediation in particular. We report a de novo-assembled sequence and annotation of the genome of a novel isolate of Paracoccus denitrificans originally sourced from coal mine tailings in India. The isolate was capable of utilizing N,N-dimethylformamide (DMF) as a source of carbon and nitrogen and therefore holds potential for bioremediation and mineralization of industrial pollutants. The genome sequence and biological circuitry revealed thereupon will be invaluable in understanding the metabolic capabilities, functioning, and evolution of this important bacterial organism.

Organophosphate Hydrolase in Brevundimonas diminuta Is Targeted to the Periplasmic Face of the Inner Membrane by the Twin Arginine Translocation Pathway

A twin arginine translocation (Tat) motif, involved in transport of folded proteins across the inner membrane, was identified in the signal peptide of the membrane-associated organophosphate hydrolase (OPH) of Brevundimonas diminuta. Expression of the precursor form of OPH carrying a C-terminal His tag in an opd-negative background and subsequent immunoblotting with anti-His antibodies showed that only the mature form of OPH associated with the membrane and that the precursor form of OPH was entirely found in the cytoplasm. When OPH was expressed without the signal peptide, most of it remained in the cytoplasm, where it was apparently correctly folded and showed activity comparable to that of the membrane-associated OPH encoded by the wild-type opd gene. Amino acid substitutions in the invariant arginine residues of the Tat signal peptide affected both the processing and localization of OPH, confirming a critical role for the Tat system in membrane targeting of OPH in B. diminuta. The localization of OPH to the periplasmic face of the inner membrane in B. diminuta was demonstrated by proteinase K treatment of spheroplasts and also by fluorescence-activated cell sorting analysis of cells expressing OPH-green fluorescent protein fusions with and without an SsrA tag that targets cytoplasmic proteins to the ClpXP protease.

Biodegradation of Aromatic Compounds : An Overview of Meta-Fission Product Hydrolases

Meta fission product (MFP) hydrolases catalyze hydrolysis of a low reactive carbon-carbon bond found in meta-fission products, generated during biodegradation of various aromatic compounds. These enzymes belong to the alpha/beta hydrolase super family and show structural conservation despite having poor sequence similarity. MFP-hydrolases are substrate specific and studies have indicated that this substrate specificity plays a key role in the determination of the organisms ability to degrade a particular substrate. In this concise review of MFP-hydrolases we discuss their classification, biochemical properties, the molecular basis of their substrate specificity, their catalytic mechanism, and evolutionary significance.

Indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonasdiminuta is self-transmissible and plays a key role in horizontal mobility of the opd gene.

A fosmid library of the 66kb indigenous organophosphate-degrading (opd) plasmid pCMS1 of Brevundimonas diminuta was tagged with mini-transposon EZTn5 , to determine its sequence using transposon-specific primers. The sequence revealed the presence of a number of tra genes suggesting their role in conjugal transfer of pCMS1. Consistent with the presence of the tra genes, the B. diminuta plasmid, pCMS1::tet, generated by replacing the opd gene with opd::tet, served as a donor for transferring pCMS1::tet into recipient strain Pseudomonas putida. The self-transmissibility of the opd-containing plasmid pCMS1 and the existence of identical opd genes on otherwise dissimilar plasmids suggests a probable role of indigenous opd plasmids like pCMS1 in transferring the opd gene among soil bacteria.

Mineralization of Acephate, a Recalcitrant Organophosphate Insecticide Is Initiated by a Pseudomonad in Environmental Samples

An aerobic bacterium capable of breaking down the pesticide acephate (O,S-dimethyl acetyl phosphoramidothioic acid) was isolated from activated sludge collected from a pesticide manufacturing facility. A phylogenetic tree based on the 16 S rRNA gene sequence determined that the isolate lies within the Pseudomonads. The isolate was able to grow in the presence of acephate at concentrations up to 80 mM, with maximum growth at 40 mM. HPLC and LC-MS/MS analysis of spent medium from growth experiments and a resting cell assay detected the accumulation of methamidophos and acetate, suggesting initial hydrolysis of the amide linkage found between these two moieties. As expected, the rapid decline in acephate was coincident with the accumulation of methamidophos. Methamidophos concentrations were maintained over a period of days, without evidence of further metabolism or cell growth by the cultures. Considering this limitation, strains such as described in this work can promote the first step of acephate mineralization in soil microbial communities.

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 σ54-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°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.

OxyR-dependent expression of a novel glutathione S-transferase (Abgst01) gene in Acinetobacter baumannii DS002 and its role in biotransformation of organophosphate insecticides

While screening a genomic library of Acinetobacter baumannii DS002 isolated from organophosphate (OP)-polluted soils, nine ORFs were identified coding for glutathione S-transferase (GST)-like proteins. These GSTs (AbGST01-AbGST09) are phylogenetically related to a number of well-characterized GST classes found in taxonomically diverse groups of organisms. Interestingly, expression of Abgst01 (GenBank accession no. KF151191) was upregulated when the bacterium was grown in the presence of an OP insecticide, methyl parathion (MeP). The gene product, AbGST01, dealkylated MeP to desMeP. An OxyR-binding motif was identified directly upstream of Abgst01. An Abgst-lacZ gene fusion lacking the OxyR-binding site showed a drastic reduction in promoter activity. Very low β-galactosidase activity levels were observed when the Abgst-lacZ fusion was mobilized into an oxyR (GenBank accession no. KF151190) null mutant of A. baumannii DS002, confirming the important role of OxyR. The OxyR-binding sites are not found upstream of other Abgst (Abgst02-Abgst09) genes. However, they contained consensus sequence motifs that can serve as possible target sites for certain well-characterized transcription factors. In support of this observation, the Abgst genes responded differentially to different oxidative stress inducers. The Abgst genes identified in A. baumannii DS002 are found to be conserved highly among all known genome sequences of A. baumannii strains. The versatile ecological adaptability of A. baumannii strains is apparent if sequence conservation is seen together with their involvement in detoxification processes.