Evolution of Phosphotriesterases (PTEs): How bacteria can acquire new degradative functions

The promiscuity of enzymes has often been considered a vestige activity based on the broad substrate spectrum of their progenitors. As such, divergent enzymes can be used as a fingerprint to track their evolutionary history. In the presence of structural mimics of active site or binding site ligands and assisted by mutations in the associated binding site, this promiscuity contributes to acquisition of new catalytic functions. This phenomenon is often referred to as substrate-assisted gain-of-function and helps soil microbes to thrive on re-calcitrant xenobiotic molecules, hitherto unfamiliar to the microbial world. This review describes the evolution of organophosphorous hydrolases, which potentially and originally functioned as quorum-sensing ‘quenching’ lactonases and highlights their remarkable horizontal mobility within diverse bacterial species

Free radical quenching activity and polyphenols in three species of Coleus

Coleus is an important aromatic herb of the family Lamiaceae which is routinely grown as a traditional medicinal herb in India. We examined the total content of polyphenols, tannins, flavones and flavonols, their antioxidant and lipid peroxidation inhibition properties in leaf and stem tissues of three species of Coleus (Coleus forskholii Briq., Coleus aromaticus Benth. and Coleus zeylanicus Benth.). Plant extracts of C. forskholii exhibited high amounts of polyphenols and higher antioxidant activity in the tissues compared to C. aromaticus and C. zeylanicus. The leaf extracts of C. forskholii showed significantly high amounts of total polyphenols (23.46 mg g-1 fw), flavones and flavonols (250.8 μg g-1 fw) and high antioxidant activity (12.29 mM g-1 fw). HPLC profiling of leaf and stem tissues showed the presence of standard antioxidative polyphenols and more potent antioxidative polyphenols. Our results demonstrate that C. forskholii could be used as an important source of phenolic compounds with significantly high antioxidant activity

Multiple mechanisms contribute to lateral transfer of an organophosphate degradation (opd) island in Sphingobium fuliginis ATCC 27551

The complete sequence of pPDL2 (37,317 bp), an indigenous plasmid of Sphingobium fuliginis ATCC 27551 that encodes genes for organophosphate degradation (opd), revealed the existence of a site-specific integrase (int) gene with an attachment site attP, typically seen in Integrative Mobilizable Elements (IME). In agreement with this sequence information, site-specific recombination was observed between pPDL2 and an artificial plasmid having a temperature-sensitive replicon and a cloned attB site at the 3′ end of the seryl tRNA gene of Sphingobium japonicum. The opd gene cluster on pPDL2 was found to be part of an active catabolic transposon with mobile elements y4qE and Tn3 at its flanking ends. Besides the previously reported opd cluster, this transposon contains genes coding for protocatechuate dioxygenase and for two transport proteins from the major facilitator family that are predicted to be involved in transport and metabolism of aromatic compounds. A pPDL2 derivative, pPDL2-K, was horizontally transferred into Escherichia coli and Acinetobacter strains, suggesting that the oriT identified in pPDL2 is functional. A well-defined replicative origin (oriV), repA was identified along with a plasmid addiction module relB/relE that would support stable maintenance of pPDL2 in Sphingobium fuliginis ATCC 27551. However, if pPDL2 is laterally transferred into hosts that do not support its replication, the opd cluster appears to integrate into the host chromosome, either through transposition or through site-specific integration. The data presented in this study help to explain the existence of identical opd genes 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

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme

Topological analysis of the lipoprotein organophosphate hydrolase from Sphingopyxis wildii reveals a periplasmic localisation

Organophosphate Hydrolase (OPH) is a membrane-associated lipoprotein. It translocates across the inner membrane via the twin-arginine transport pathway and remains anchored to the periplasmic face of the inner membrane through a diacylglycerol moiety linked to the invariant cysteine residue found at the junction of a SpaseII cleavage site. Due to the existence of a transmembrane helix at the C-terminus of the mature OPH, an inner-membrane topology was predicted suggesting the C-terminus of OPH is cytoplasmic. The predicted topology was validated by generating OPH variants either fused in-frame with β-lactamase or with unique cysteine residues. Sphingopyxis wildii cells expressing OPH variants with Bla fused at the N-terminal, C-terminal or central regions all grew in the presence of ampicillin. Supporting the β-lactamase reporter assay, the OPH variants having unique cysteine residues at different strategic locations were accessible to the otherwise membrane-impermeant PEG-Mal (methoxypolyethylene glycol maleimide) revealing that, with the exception of the lipoprotein anchor, the entire OPH is in the periplasmic space

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 nijB and nijF 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 λp_Lp_R) 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 SIDS-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

Purification and characterization of catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and cloning, sequencing of partial catA gene

Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent Km and Vmax for intradiol cleavage of catechol were 1.58 μM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609)

Self-transmissible nif Plasmid (pEA9) of Enterobacter agglomerans339: 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 agglomerans339. 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 agglomerans333. 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

A fluorescence based assay with pyranine labeled hexa-histidine tagged Organophosphorus hydrolase (OPH) for determination of organophosphates

Organophosphorus hydrolase has been employed extensively for catalysis based biosensor synthesis with optical, potentiometric and amperometric based detection mechanism. The present study reports the response of bioconjuagtes prepared by utilizing OPH and pH sensitive polyanionic fluorophore i.e. pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) in different molar ratios by the aid of electrostatic force. Two types of bioconjugates were prepared, one of them contained mature form of OPH obtained from Escherichia coli cells expressing organophosphate degrading (opd) gene from a tac promoter, whereas the variant OPH6His, has 6× histidine tail at C-terminus. The investigation was carried out utilizing the bioconjugates of different molar ratios prepared with both the enzymes and ratio showing maximum degree of labeling with pyranine was selected for the detection of organophosphates in standard samples. The lower limit of detection for paraoxon was ∼20 ppb and for methyl parathion and coumaphos it was ∼50 ppb under optimized conditions (55–60 °C) with the reaction time of 3 min. These features of the prepared conjugate make it a strong contender for the development of the field deployable biosensor for organophosphates estimation