Metagenomic Characterization of Bacterial Communities in Drinking Water Supply System of a Mega City.

Supplying safe water to consumers is vital for protection of public health. With population of > 15 million, Karachi is the main economical hub of Pakistan. Lake Keenjhar serves as the main source of fresh water while Hub dam is the secondary water reservoir for Karachi. In this study, bacterial community of the drinking water supply system (DWSS) of Karachi was studied from source to tap using metagenomics approach. For this purpose, we collected 41 water samples from different areas of the city (n = 38) and water reservoirs (n = 3). 16S rDNA metagenomic sequencing of water samples revealed that 88% sequences were associated with Proteobacteria (52%), Planctomycetes (15%), Becteroidetes (12%), and Verrucomicrobia (6%). On the class level, α-proteobacteria (6-56%) were found to be the most abundant followed by ß- (8-41%) and γ-proteobacteria (6-52%). On the genus level, substantial diversity was observed among the samples. Bacterial communities in water from Hub dam was found to be distantly related while among the residential towns, Lyari was highly distant from the others. Twenty-four bacterial genera were found to be exclusively present in residential area samples in comparison to the source waters which is suggestive of their resistance against treatment procedures and/or contamination. Metagenomic analysis revealed abundance of Pseudomonas, Legionella, Neisseria, Acinetobacter, Bosea, and Microcystis genera in residential areas water samples. The present metagenomic analysis of DWSS of Karachi has allowed the evaluation of bacterial communities in source water and the water being supplied to the city. Moreover, measurement of heavy metals in water samples from Karachi revealed arsenic concentration according to WHO standards which is in contrast of recent study which reported extensive arsenic contamination in aquifers in the Indus valley plain. To the best of our knowledge, this is the first metagenomic study of DWSS of Karachi.

Small molecule activators of proteasome-related HslV peptidase.

The HslVU is the proteasome-related two component system composed of HslV peptidase and HslU chaperone. It is involved in the degradation of an array of intracellular proteins. The presence of HslVU homologs in pathogenic microbes and its absence in human makes it an antimicrobial drug target. The functional HslVU complex forms when HslV dodecamer is flanked at both ends by HslU hexamers. In the HslVU complex, eight residues at the carboxy termini of HslU subunits intercalate into a clefts between two adjacent HslV subunits causing a conformational change in the active site of HslV which in turn results in the allosteric activation of HslV peptidase. Here, we report small molecules capable of activating HslV peptidase in the absence of its natural activator HslU ATPase. For this purpose, virtual screening of an in-house library of synthetic and natural compounds was performed to find out ligands mimicking the interaction of HslU carboxy terminus with HslV dodecamer. The benzimidazole, quinazoline and chromone derivatives were suggested by ligand docking to bind at the HslU carboxy termini intercalation pockets in the HslV dodecamer. This was confirmed by HslV activation and isothermal titration calorimetry assays with these compounds that gave ED(50) in sub-micromolar range (0.6-1.5µM). The results showed for the first time that small, extracellular non-peptidic molecules can allosterically activate the peptide hydrolytic activity of HslV which in turn would initiate intracellular proteolysis.

Structural bioinformatics of Neisseria meningitidis LD-carboxypeptidase: implications for substrate binding and specificity.

Neisseria meningitidis, a gram negative bacterium, is the leading cause of bacterial meningitis and severe sepsis. Neisseria meningitidis genome contains 2,160 predicted coding regions including 1,000 hypothetical genes. Re-annotation of N. meningitidis hypothetical proteins identified nine putative peptidases. Among them, the NMB1620 protein was annotated as LD-carboxypeptidase involved in peptidoglycan recycling. Structural bioinformatics studies of NMB1620 protein using homology modeling and ligand docking were carried out. Structural comparison of substrate binding site of LD-carboxypeptidase was performed based on binding of tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'. Inspection of different subsite-forming residues showed changeability in the S1 subsite across different bacterial species. This variability was predicted to provide a structural basis to S1-subsite for accommodating different amino acid residues at P1 position of the tetrapeptide substrate 'L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanine'.