Identification of arsenic oxidizing genes fragment in Microbacterium sp. strain 1S1 and its cloning in E. coli (DH5a).

Microbacterium sp. strain 1S1, an arsenic-resistant bacterial strain, was isolated with 75 mM MIC against arsenite. Brownish precipitation with silver nitrate appeared, which confirmed its oxidizing ability against arsenite. The bacterial genomic DNA underwent Illumina and Nanopore sequencing, revealing a distinctive cluster of genes spanning 9.6 kb associated with arsenite oxidation. These genes were identified within an isolated bacterial strain. Notably, the smaller subunit (aioB) of the arsenite oxidizing gene at the chromosomal DNA locus (Prokka_01508) was pinpointed. This gene, aioB, is pivotal in arsenite oxidation, a process crucial for energy metabolism. Upon thorough sequencing analysis, only a singular megaplasmid was detected within the isolated bacterial strain. Strikingly, this megaplasmid did not harbor any genes responsible for arsenic resistance or detoxification. This intriguingly indicates that the bacterial strain relies on the arsenic oxidizing genes present for its efficient arsenic oxidation capability. This is especially true for Microbacterium sp. strain 1S1. Subsequently, a segment of genes linked to arsenic resistance was successfully cloned into E. coli (DH5a). The fragment of arsenic-resistant genes was cloned in E. coli (DH5a), further confirmed by the AgNO3 method. This genetically engineered E. coli (DH5a) can decontaminate arsenic-contaminated sites.

Characterization of plastic degrading bacteria isolated from sewage wastewater.

Plastic is a fundamental polymer used in routine life and disposed of in sewage. It leads to microplastic pollution in aquatic organisms, introducing it into the food chain and affecting human health. In the present study, samples were collected from sewage wastewater to isolate the bacteria that could potentially reduce plastic. The six samples were incubated with plastic pieces in minimal salt media for 120 days. After 120 days, the weight loss experiment showed that samples SH5B and SH6B degraded 25% plastic. After chemical and molecular characterization, these strains were identified as Pseudomonas sp. SH5B and Pseudomonas aeruginosa SH6B. The Fourier-transform infrared spectroscopy (FTIR) analysis showed peaks shifting, indicating bond stretching, bond bending, and new bond formation. The Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed various new compounds produced during plastic degradation by these bacterial strains. The plastic biodegradation potential makes these bacteria an impending foundation for green chemistry to eradicate tough pollutants from the environment.

Biotoxicity comparison of Bacillus thuringiensis to control vector borne diseases against mosquito fauna.

The current study was designed to evaluate the biotoxicity of screened echo-friendly Bacillus thuringiensis strains from different areas of Pakistan. Out of 50 samples, 36% Bt. isolates were quarantined from soil containing cattle waste after morphological, biochemical, and molecular characterization. The toxicity bioassays with Bt. spores and protein diet proved that 11 Bt. isolates were utmost noxious to 3rd instar larvae of mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The entopathogenic activity of first 4 Bt. toxins against A. aegypti was highly lethal as compared to the other dipteran larvae. The toxicity (LC50) of spore diet of Bt. strains GCU-DAB-NF4 (442.730 ± 0.38 µg/ml), NF6 (460.845 ± 0.29 µg/ml), NF3 (470.129 ± 0.28 µg/ml), and NF7 (493.637 ± 0.70 µg/ml) was quite high against A. aegypti as compared to the C. pipiens after 24 h of incubation. The highest toxicity of total cell protein was shown by GCU-DAB-NF4 (LC50 = 84.10 ± 50 µg/ml), NF6 (95.122 ± 0.40 µg/ml), NF3 (100.715 ± 06 µg/ml), and NF5 (103.40 ± 07 µg/ml) against A. aegypti after 24 h. So, these strains a have great potential to be used as biological control especially against A. aegypti as compared to the C. pipiens.

Biotoxicity assessment of cloned cry 11 protein gene from Bacillus thuringiensis 9NF.

The current investigation describes the isolation and characterization of toxic Bt. local isolates harboring 99% homology with Bti. prototoxin Bacillus thuringiensis (AXJ97553.1 and novel OUB27301.1) which contains full length cry11 gene (1.9 kb). Initially, it was cloned in pTZ57R/T and then sub-cloned in pET30a(+) for expression. The optimized conditions for good expression were found 1 mM IPTG, 3.5-4 h incubation time, and 37 °C. Toxicological assays were determined against 3rd instar larvae of Aedes aegypti with expressed partially purified and crude recombinant protein using recombinant E. coli BL21, DE3 transformed with cry11 gene. It was found that partially purified Bt. protein is highly toxic against A. aegypti larvae with LC50 value of 42.883 ± 6 µg/ml. B. thuringiensis strains producing Cry 11 toxic protein can be used as biopesticide to control resistance in insects.