Putative Role of Anti-microbial Peptide Recovered from Lactiplantibacillus spp. in Biocontrol Activity.

Antimicrobial peptides (AMPs) stand as a promising alternative to conventional pesticides, leveraging a multifaceted approach to combat plant pathogens. This study focuses on identifying and characterizing the AMP produced by Lactiplantibacillus argentoratensis strain IT, demonstrating potent antibacterial activity against various harmful microorganisms. Evaluation of AMPs' antibacterial activity was conducted through an agar well diffusion assay, a reliable method for assessing secondary metabolite antimicrobial efficacy. The study unveils the antimicrobial potential of the purified extract obtained from Lactiplantibacillus argentoratensis IT, isolated from goat milk. Notably, the AMP exhibited robust antibacterial activity against phytopathogens affecting solanaceous crops, including the Gram-negative Ralstonia solanacearum. Expression conditions and purification methods were optimized to identify the peptide's mass and sequence, utilizing LC-MS and SDS-PAGE. This paper underscores the application potential of Lactiplantibacillus spp. IT as a biocontrol agent for managing bacterial infectious diseases in plants. Results indicate optimal AMP production at 37 °C, with a culture broth pH of 5 during fermentation. The obtained peptide sequence corresponded to peaks at 842.5 and 2866.4 m/z ratio, with a molecular weight of approximately 5 kDa according to tricine SDS-PAGE analysis. In conclusion, this study lays the foundation for utilizing Lactiplantibacillus spp. IT derived AMPs in plant biocontrol strategies, showcasing their efficacy against bacterial phytopathogens. These findings contribute valuable insights for advancing sustainable agricultural practices.

Isolation and Characterization of a Novel Laccase-Producing Bacteria Bhargavaea beijingensis from Paper and Pulp Effluent-Treated Soil Using In Silico Approaches.

Laccases (EC 1.10.3.2) are considered one of the most prominent multicopper enzymes that exhibit the inherent properties of oxidizing a range of phenolic substrates. Mostly, reported laccases have been isolated from the plants and fungi species, whereas bacterial laccases are yet to be explored. Bacterial laccases have numerous distinctive properties over fungal laccases, including stability at high temperatures and high pH. This study includes the isolation of bacteria through the soil sample collected from the paper and pulp industry; the highest laccase-producing bacteria was identified as Bhargavaea bejingensis, using 16S rRNA gene sequencing. The extracellular and intracellular activities after 24 h incubation were 1.41 U/mL and 4.95 U/mL, respectively. The laccase-encoding gene of the bacteria was sequenced; moreover, the in vitro translated protein was bioinformatically characterized and asserted that the laccase produced by the bacteria Bhargavaea bejingensis was structurally and sequentially homologous to the CotA protein of Bacillus subtilis. The enzyme laccase produced from B. bejingensis was classified as three-domain laccase with several copper-binding residues, where a few crucial copper-binding residues of the laccase enzyme were also predicted.

Bioprospecting uncultivable microbial diversity in tannery effluent contaminated soil using shotgun sequencing and bio-reduction of chromium by indigenous chromate reductase genes.

The tannery industry generates a consequential threat to the environment by producing a large amount of potentially toxic metal-containing waste. Bioremediation has been a promising approach for treating potentially toxic metals, but the efficiency of remediation in microbes is one of the factors limiting their application in tanneries waste treatment. The motivation behind the present work was to explore the microbial diversity and chromate reductase genes present in the tannery effluent-contaminated soil using metagenomics approach. The use of shotgun sequencing enabled the identification of operational parameters that influence microbiome composition and their ability to reduce Chromium (Cr) concentration. The Cr concentration in Kanpur tannery effluent contaminated soil sample was 700 ppm which is many folds than the approved permissible limit by World Health Organisation (WHO) for Cr is 100 ppm. Metagenomic Deoxyribo Nucleic Acid (DNA) was extracted to explore taxonomic community structure, phylogenetic linkages, and functional profile. With a Guanine-Cytosine (GC) abundance of 54%, total of 45,163,604 high-quality filtered reads were obtained. Bacteria (83%), Archaebacteria (14%), and Viruses (3%) were discovered in the structural biodiversity. Bacteria were classified to phylum level, with Proteobacteria (52%) being the dominant population, followed by Bacteriodetes (15%), Chloroflexi (15%), Spirochaetes (7%), Thermotogae (5%), Actinobacteria (4%), and Firmicutes (1%). The OXR genes were cloned and checked for their efficiency to reduce Cr concentration. Insitu validation of OXR8 gene showed a reduction of Cr concentration from 700 ppm to 24 ppm in 72 h (96.51% reduction). The results of this study suggests that there is a huge reservoir of microbes and chromate reductase genes which are unexplored yet.

Cloning of merA Gene from Methylotenera Mobilis for Mercury Biotransformation.

Mercury (Hg) is one of the most toxic heavy metal and is extremely harmful for the environment. The permissible limit of mercury in industrial effluents is 0.001 ppm, whereas there are various sites having very high levels of mercury contamination. In the present study, 10 different mercury (Hg) resistant bacterial strains were isolated from Ulhas Estuary, Mumbai (Hg concentration of 107 ppm). All the strains were subsequently grown on higher concentration of mercuric chloride (HgCl2), one of the isolate (USP5) showed significant growth at high concentration of Hg (40 ppm) and 16S rRNA gene sequencing revealed the identity of the bacterium as Methylotenera mobilis, (Accession no. KT714144). The mer operon was isolated and cloned in E.coli and checked for its ability to tolerate higher concentration of Hg. It has shown growth up to 70 ppm of Hg, also presence of merA gene indicated its ability to detoxify Hg into less toxic volatile form. The atomic absorption spectrophotometry confirmed the ability of clone to efficiently detoxify 60-90 % of the Hg (10-70 ppm) within 48-72 h. This clone can be used for effective volatilization of Hg from contaminated areas.

Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus.

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed.

Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches.

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process.

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H(2)) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H(2) constituted 63-69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34-38 mm) -Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56-62 mm) -Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) -B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H(2) producing abilities in the range of 0.26-0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) -B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H(2) - 0.63 mol/mol of glucose added and PHB - 420-435 mg/l medium.