Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of <i>Chelatococcus</i> sp.

<div><p>Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium <i>Chelatococcus</i> sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by <i>Chelatococcus</i> sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by <i>Chelatococcus</i> sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by <i>Chelatococcus</i> sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy –3, 3^/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that <i>Chelatococcus</i> sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of <i>Chelatococcus</i> sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of <i>Chelatococcus</i> sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that <i>Chelatococcus</i> sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.</p></div

Bacterial growth and protein content of culture medium inoculated with <i>Chelatococcus</i> sp. in presence or absence of DBT (0.5 mM).

<p>A: growth in presence of DBT, B: growth in absence of DBT. Values are mean ± S.D of triplicate determinations. Significance of difference with respect to growth of bacteria in presence of 0.5 mM DBT.</p

GC-MS analysis of DBT metabolites produced by C<i>helatococcus</i> sp.

<p><b>A</b>: GC profile of the culture extract (96 h post inoculation) showing formation of 2- HBP, 2-MBP, and DBTO; <b>B</b>: Mass spectrum of 2-HBP (molecular mass, 170); <b>C</b>: Mass spectrum of 2-MBP (molecular mass, 184).</p

GC–FID chromatogram of desulphurization of diesel oil by <i>Chelatococcus</i> sp.

<p><b>(A)</b> Control diesel fuel; <b>(B)</b> Diesel fuel treated with C<i>helatococcus</i> sp. for 24 h at 37°C.</p

Phylogenetic relationships of isolated NBTU-06 and other closely related <i>Chelatococcus</i> species based on 16S rRNA sequencing.

<p>The tree was generated using the neighbor-joining method and the sequence from <i>Bacillus</i> sp. HSCC 1649 T (<i>Accession no</i>. <i>AB045097</i>) was considered as out-group. The data set was resampled 1,000 times by using the bootstrap option, and percentage values are given at the nodes.</p

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

AbstractAn acidic phospholipase A2 (RVVA-PLA2-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA2-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA2 enzyme. RVVA-PLA2-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA2-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA2-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA2s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA2-I suggested the correlation between catalytic and membrane damaging activities of this PLA2 enzyme. Our study advocates that the presence of a large number of PLA2-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA2 enzyme

GC-MS analysis of metabolites of 4-M- DBT produced by <i>Chelatococcus</i> sp.

<p><b>A</b>: GC chromatogram of the culture extract showing DBTO, 2-hydroxy-3’ methyl–biphenyl and 4-M- DBT; <b>B</b>: Mass spectrum of DBTO (molecular mass, 200); <b>C</b>: Mass spectrum of 2-hydroxy-3’ methyl—biphenyl (molecular mass, 184); <b>D</b>: Mass spectrum of 4-M- DBT (molecular mass, 198).</p

Kinetics of utilization of DBT by <i>Chelatococcus</i> sp. in liquid BSM as the sole source of sulfur.

<p>Residual DBT concentration was determined by RP-HPLC analysis of extract from the culture medium (⬛) inoculated with <i>Chelatococcus</i> sp. and uninoculated controls (▲). Values are mean ± SD of triplicate determinations.</p

GC-MS analysis of metabolites of 4, 6 -DM- DBT produced by <i>Chelatococcus</i> sp.

<p><b>A</b>: GC chromatogram of the culture extract showing, 2-hydroxy-3, 3’ dimethyl–biphenyl and 4, 6 -M- DBT; <b>B</b>: Mass spectrum of 2-hydroxy-3, 3’ dimethyl—biphenyl (molecular mass, 198); <b>C</b>: Mass spectrum of 4, 6-DM- DBT (molecular mass, 212).</p

A scheme for extended 4s pathway of biocatalytic desulfurization of DBT by <i>Chelatococcus</i> sp.

<p>A scheme for extended 4s pathway of biocatalytic desulfurization of DBT by <i>Chelatococcus</i> sp.</p