Pw2

This study characterized two novel Siphoviridae phages, PW2 and PW4, that can infect 52% and 44% of the tested Bacillus cereus group isolates and display relatively high activity against four cereulide-producing isolates belonging to B. weihenstephanensis and B. paranthracis. The genome sequences of PW2 and PW4 are similar to six known phages infecting B. cereus group isolates, which can be classified into two conserved groups, with the PW2 genome harboring conserved coding sequences (CDSs) from both groups. Two phage-derived endolysins, LysPW2 and LysPW4, which are predicted to encode N-acetylmuramoyl-L-alanine amidase, and their enzymatically active domains (EADs), LysPW2-EAD and LysPW4-EAD, were heterologously expressed. Both LysPW2 and LysPW4, especially the former, show a much wider host range than the phages, albeit still limited to the B. cereus group for the tested bacteria. The optimal temperature and pH for LysPW2 ability is 37 °C and pH 8.0 and for LysPW4 is 50 °C and pH 9.0. Neither LysPW2-EAD nor LysPW4-EAD show any lytic activity against vegetative cells of the tested B. cereus group isolates but can inhibit germination in 66.3% and 65.7% of spores, respectively. In addition, both LysPW2-EAD and LysPW4-EAD exhibit spore-binding capabilities.

Oxytetracycline (OTC), is a widely used veterinary antibiotic for treatment and prophylaxis in aquaculture. As an emerging pollutant, OTC in the environment exerts selective pressure on aquatic organisms causing proliferation of antibiotic resistant genes. In the present study, an OTC tolerant isolate labelled as pw2 was selected among the 11 OTC tolerant isolates, isolated from the aquaculture effluent, for investigating its OTC degrading potential. The cell morphology, biochemical characteristics, and 16S ribosomal RNA (rRNA) gene sequence of the isolated strain indicated that it belonged to the genus Planococcus. The OTC removal percentage was estimated through measuring its residual concentration in the culture medium with high performance liquid chromatography. The strain exhibited maximum removal efficiency of 90.62%, with initial OTC concentration of 10 µg/ml. The optimum degrading conditions were 35 °C and pH 7. The degradation rate of OTC with (biotic) and without strain pw2 (abiotic) was 3.253 and 1.149 mg/l/d, respectively. The half-life was recorded to be 2.13 d in the presence of strain pw2, in contrast to 6.03 days recorded without strain pw2. The total (biotic + abiotic) OTC degradation efficiency was 75.74, 83.93, 90.62, and 86.47% for the initial OTC concentrations of 1 to 25 µg/ml, respectively. Addition of carbon and nitrogen did not influence the OTC removal which indicates Planococcus sp. pw2 use OTC as sole energy source. Thus, Planococcus sp. pw2 plays a vital role in reducing the OTC concentration in the environment, offering a promising method for treatment of aquaculture effluent containing OTC.

Background: Thermostable cellulases are in constant demand for several biotechnological applications. Two thermophilic bacterial strains PW1 and PW2 isolated from Tattapani hot spring were found to have cellulolytic activity. Subsequently, PW1 and PW2 were identified and mined for genes encoding cellulase activity. Results: Sequencing of the 16S rDNA of PW1 and PW2 identified them as Bacillus sp. PW1 (Acc no. KU711837) and Bacillus sp. PW2 (Acc no. KU711838), respectively, which clustered in the clades containing thermophilic members of Bacillus sp. and Geobacillus species. Phylogenetic analysis revealed that despite the morphological and sequence identities, Bacillus sp. PW1 and Bacillus sp. PW2 are different at the genetic level. The cellulase genes (~ 1.1 kb) of the two bacterial strains were amplified using primers designed against related thermophilic cellulases. Sequencing of the cellulase gene amplicons of PW1 and PW2 revealed that they encode proteins of 280 and 206 amino acid residues, respectively. Sequence and domain analysis of the protein products of PW1 and PW2 revealed that they belong to M42 family of aminopeptidase/endoglucanase. The PW2 endoglucanase coding sequence was submitted to Genbank under accession no. MH049504. The structures of putative endoglucanases of PW1 and PW2 were generated using 1VHE.A as template, which showed the presence of vast proportion of random coils. Molecular docking of the modeled endoglucanase proteins with various substrates and products of cellulases showed that carboxymethyl cellulose and maltose exhibit the highest binding affinity, while xylan and glucose the least. Conclusions: The two thermophilic bacteria PW1 and PW2 and their endoglucanase gene can be further utilized for recombinant production of thermostable cellulases for their application in industries.