Whole-Genome Sequencing and Annotation of Bacillus safensis RIT372 and Pseudomonas oryzihabitans RIT370 from Capsicum annuum (Bird's Eye Chili) and Capsicum chinense (Yellow Lantern Chili), Respectively

The plant genus Capsium (Solanaceae) is of interest given its use in a number of herbal remedies by indigenous inhabitants throughout the world and the ability to synthesize the antibacterial metabolite and the active ingredient capsaicin (8-methyl-N-vanillyl-6-nonenamide) which is predominantly stored in the fruit of the plant (1, 2). To gain some insights into bacteria that associate with the Capsicum spp., we embarked on a project as a laboratory exercise in the Fundamentals of Plant Biochemistry and Pathology course in the Biotechnology and Molecular Bioscience Program at the Rochester Institute of Technology to assess the bacterial diversity of bacteria associate with the fruits of Capsicum annuum (bird's eye chili) and Capsicum chinense (yellow lantern chili). This resulted in the isolation and identification of Bacillus safensis RIT372 and Pseudomonas oryzihabitans RIT370 from surface sterilized internal tissue from the fruits that were purchased from a farmer's market in Rochester, New York. The bacteria were initially identified as the Bacillus sp. and Pseudomonas sp. through the amplification and nucleotide sequence analysis of the variable 3 (V3) region from the 16S rRNA gene using the primers V3-forward 5′-ACTCCTACGGGAGGCAGCAG-3′ and V3-reverse 5′-ATTACCGCGGCTGCTGG-3 (3).

Genomic DNA (gDNA) from RIT372 and RIT370 was isolated from the bacteria using the GenElute bacterial genomic kit (Sigma-Aldrich, St. Louis, MO). The gDNA was prepared for whole-genome sequencing using a Nextera XT library preparation kit (Illumina, San Diego, CA). Whole-genome sequencing was performed using the Illumina Miseq (250-bp paired-end reads). The reads were error-corrected and assembled de novo using Spades 2.5 (4). The contigs were submitted to the RAST server for annotation. Genome-based taxonomic assignment of RIT372 and RIT370 was performed using JSpecies with an average nucleotide identity (ANI) cutoff value of >95% for species delineation (5). For RIT372, the de novo assembly and RAST annotation resulted in a genome size of 3,676,140 bp (38× coverage, 41.73% G+C content, N₅₀ 70,686 bp, and 93 contigs), 3,787 open reading frames (ORFs), and 82 RNAs and for RIT370, 5,092,972 bp (30× coverage, 66.00% G+C content, N₅₀ of 222,589 bp, 47 contigs), 66 RNAs, and 4,600 ORFs.

Secondary metabolite analysis of RIT372 and RIT370 using the antibiotics and secondary metabolite analysis shell (antiSMASH) tool resulted in the identification of gene clusters for the synthesis of bacilysin, where 60% of genes show homology in contig 2 between nucleotides 110,328 and 151,758, lichenysin, where 92% of genes show homology in contig 10 between nucleotides 8,808 and 92,529, bacillibactin, where 53% of genes show homology in contig 13 between nucleotides 10,936 and 60,646, sporulation killing factor, where 71% of genes show homology in contig 17 between nucleotides 59,844 and 77,860 for RIT372 and gene clusters for the synthesis of the siderophore turnerbactin, where 60% of genes show homology in contig 1 between nucleotides 158,226 and 211,070, and the terpene carotenoid, where 100% of genes show homology in contig 6 between nucleotides 41,781 and 65,405 for RIT370 (6, 7).

This work was supported by the College of Science and the Thomas H. Gosnell School of Life Sciences at the Rochester Institute of Technology (RIT) in addition to support from the Monash University Malaysia Tropical Medicine and Biology Multidisciplinary Platform. K.D.M. and A.A.C. were funded through the Louis Stokes Alliance for Minority Participation (LSAMP) through the National Science Foundation (HRD-1202480).