Phenotypic characterization and genome analysis reveal the probiotic potential of a banyan endophyte Bacillus velezensis K1.

AIM: The current study aimed to scrutinize the probiotic traits and safety aspects of Bacillus velezensis K1 through experimental and supporting genome studies. METHODS AND RESULTS: The seven cultures previously isolated from the aerial roots of Ficus benghalensis were initially screened for their antibacterial activity as well as acid and bile tolerance. The isolate K1 was found to be the most potent and was further investigated for probiotic traits and safety. K1 showed tolerance to simulated digestive juices and 0.3% bile. It showed notable aggregation, cell surface hydrophobicity, and adherence to HT-29 cells. K1 significantly prevented the adhesion of E. coli O157: H7 and S. enterica ATCC 13076 to HT-29 in cell culture assays. K1 could hydrolyze phytate and complex polysaccharides. The genes related to stress tolerance, adhesion, antimicrobial activity, and production of vitamins, viz. thiamine, riboflavin, pyridoxine, pantothenic acid, folate, and biotin were annotated in the K1 genome. K1 was found to be non-hemolytic, noncytotoxic, as well as susceptible to antibiotics. No virulence or toxin-encoding genes were identified in its genome. CONCLUSIONS: B. velezensis K1 is a prospective probiotic with the ability to tolerate gastrointestinal stress, adhere to intestinal surfaces, and inhibit enteropathogens.

Genome analysis uncovers the prolific antagonistic and plant growth-promoting potential of endophyte Bacillus velezensis K1.

Insights into the application of endophytic bacilli in sustainable agricultural practices have opened up new avenues for the inhibition of soil-borne pathogens and the improvement of plant health. Bacillus subtilis K1, an endophytic bacterium originally isolated from aerial roots of Ficus benghalensis is a potential biocontrol agent secreting a mixture of surfactins, iturins and fengycins. The current study extends the characterization of this bacterium through genomic and comparative genomics approaches. The sequencing of the bacterial genome at Illumina MiSeq platform revealed that it possessed a 4,103,502-bp circular chromosome with 45.98% GC content and 4325 predicted protein-coding sequences. Based on phylogenomics and whole-genome average nucleotide identity, the B. subtilis K1 was taxonomically classified as Bacillus velezensis. The formerly evaluated phenotypic traits viz. C-source utilization and lipopeptide-mediated fungal antagonism were correlated to their molecular determinants. The genome also harbored several genes associated with induced systemic resistance and plant growth promotion i.e, phytohormone production, nitrogen assimilation and reduction, siderophore production, phosphate solubilization, biofilm formation, swarming motility, acetoin and butanediol synthesis. The production of antifungal volatile organic compounds and plant growth promotion was experimentally demonstrated by volatile compound assay and seed germination assay on cumin and groundnut. The isolate also holds great prospects for application as a soil inoculant as indicated by enhancement in the growth of groundnut via in planta pot studies. Bacterial pan-genome analysis based on a comparison of whole genomes with eighteen other Bacillus strains was also conducted. Comparative examination of biosynthetic gene clusters across all genomes indicated that the largest number of gene clusters were harbored by the K1 genome. Based on the findings, we propose K1 as a model for scrutinizing non-ribosomally synthesized peptide synthetase and polyketide synthetase derived molecules.

Genome assisted probiotic characterization and application of Bacillus velezensis ZBG17 as an alternative to antibiotic growth promoters in broiler chickens.

The present study describes genome annotation and phenotypic characterization of Bacillus velezensis ZBG17 and evaluation of its performance as antibiotic growth promoter substitute in broiler chickens. ZBG17 comprises 3.89 Mbp genome with GC content of 46.5%. ZBG17 could tolerate simulated gastrointestinal juices prevalent in the animal gut. Some adhesion-associated genomic features of ZBG17 supported the experimentally determined cell surface hydrophobicity and cell aggregation results. ZBG17 encoded multiple secondary metabolite gene clusters correlating with its broad-spectrum antibacterial activity. Interestingly, ZBG17 completely inhibited Salmonella enterica and Escherichia coli within 6 h and 8 h in liquid co-culture assay, respectively. ZBG17 genome analysis did not reveal any genetic determinant associated with reported safety hazards for use as a poultry direct-fed microbial. Dietary supplementation of ZBG17 significantly improved feed utilization efficiency and humoral immune response in broiler chickens, suggesting its prospective application as a direct-fed microbial in broiler chickens.

Phytostimulation and biocontrol potential of Gram-positive endospore-forming Bacilli.

MAIN CONCLUSION: The spore-forming Bacillus and Paenibacillus species represent the phyla of beneficial bacteria for application as agricultural inputs in form of effective phytostimulators, biofertilizers, and biocontrol agents. The members of the genera Bacillus and Paenibacillus isolated from several ecological habitats are been thoroughly dissected for their effective application in the development of sustainable and eco-friendly agriculture. Numerous Bacillus and Paenibacillus species are reported as plant growth-promoting bacteria influencing the health and productivity of the food crops. This review narrates the mechanisms utilized by these species to enhance bioavailability and/or facilitate the acquisition of nutrients by the host plant, modulate plant hormones, stimulate host defense and stress resistance mechanisms, exert antagonistic action against soil and airborne pathogens, and alleviate the plant health. The mechanisms employed by Bacillus and Paenibacillus are seldom mutually exclusive. The comprehensive and systematic exploration of the aforementioned mechanisms in conjunction with the field investigations may assist in the exploration and selection of an effective biofertilizer and a biocontrol agent. This review aims to gather and discuss the literature citing the applications of Bacillus and Paenibacillus in the management of sustainable agriculture.

Genomics assisted functional characterization of Paenibacillus polymyxa HK4 as a biocontrol and plant growth promoting bacterium.

The diseases caused by phytopathogens account for huge economic losses in the agricultural sector. Paenibacillus polymyxa is one of the agriculturally important biocontrol agents and plant growth promoting bacterium. This study describes the antifungal potential of P. polymyxa HK4 against an array of fungal phytopathogens and its ability to stimulate seed germination of cumin and groundnut under in vitro conditions. The cumin and groundnut seeds bacterized with HK4 exhibited enhanced germination efficiency in comparison to controls. The use of HK4 as a soil inoculant significantly promoted the shoot length and fresh weight of groundnut plants in pot studies. The draft genome analysis of HK4 revealed the genetic attributes for motility, root colonization, antagonism, phosphate solubilization, siderophore production and production of volatile organic compounds. The bacterium HK4 harnessed several hydrolytic enzymes that may assist its competence in the rhizosphere. The PCR amplification and sequence analysis of the conserved region of the fusA gene amplicon revealed the ability of HK4 to produce fusaricidin. Furthermore, the LC-ESI-MS/MS of crude cell pellet extract of HK4 confirmed the presence of fusaricidin as a major antifungal metabolite. This study demonstrated the potential of HK4 as a biocontrol agent and a plant growth promoter.

Genome analysis reveals probiotic propensities of Paenibacillus polymyxa HK4.

The legislations on the usage of antibiotics as growth promoters and prophylactic agents have compelled to develop alternative tools to upsurge the animal protection and contain antibiotic usage. Probiotics have emerged as an effective antibiotic substitute in animal farming. The present study explores the probiotic perspective of Paenibacillus polymyxa HK4 interlinking the genotypic and phenotypic characteristics. The draft genome of HK4 revealed the presence of ORFs encoding the functions associated with tolerance to gastrointestinal stress and adhesion. The biosynthetic gene clusters encoding non-ribosomally synthesized peptides, polyketides and lanthipeptides such as fusaricidin, tridecaptin, polymyxin, paenilan and paenibacillin were annotated in HK4 genome. The strain harbored the chromosomal gene conferring the resistance to lincosamides. No functional gene encoding virulence or toxins could be identified in the genome of HK4. The genome analysis data was complemented by the in vitro experiments confirming its survival during gastrointestinal transit, antimicrobial potential and antibiotic sensitivity. NUCLEOTIDE SEQUENCE ACCESSION NUMBER: The draft-genome sequence of Paenibacillus polymyxa HK4 has been deposited as whole-genome shotgun project at GenBank under the accession number PRJNA603023.