Comparative genomics of cocci-shaped Sporosarcina strains with diverse spatial isolation

Abstract

Background: Cocci-shaped Sporosarcina strains are currently one of the few known cocci-shaped spore-forming bacteria, yet we know very little about the genomics. The goal of this study is to utilize comparative genomics to investigate the diversity of cocci-shaped Sporosarcina strains that differ in their geographical isolation and show different nutritional requirements.

Results: For this study, we sequenced 28 genomes of cocci-shaped Sporosarcina strains isolated from 13 different locations around the world. We generated the first six complete genomes and methylomes utilizing PacBio sequencing, and an additional 22 draft genomes using Illumina sequencing. Genomic analysis revealed that cocci-shaped Sporosarcina strains contained an average genome of 3.3 Mb comprised of 3222 CDS, 54 tRNAs and 6 rRNAs, while only two strains contained plasmids. The cocci-shaped Sporosarcina genome on average contained 2.3 prophages and 15.6 IS elements, while methylome analysis supported the diversity of these strains as only one of 31 methylation motifs were shared under identical growth conditions. Analysis with a 90% identity cut-off revealed 221 core genes or ~ 7% of the genome, while a 30% identity cut-off generated a pan-genome of 8610 genes. The phylogenetic relationship of the cocci-shaped Sporosarcina strains based on either core genes, accessory genes or spore-related genes consistently resulted in the 29 strains being divided into eight clades.

Conclusions: This study begins to unravel the phylogenetic relationship of cocci-shaped Sporosarcina strains, and the comparative genomics of these strains supports identification of several new species.

Keywords: Cocci; Comparative genomics; Spore-forming; Sporosarcina; Sporosarcina ureae.

Fig. 1

Geographic distribution of the genus Sporosarcina, using location data from the Earth Microbiome Project (circles), and Genbank (triangles). Colors indicate the general source of isolation, based on sequence metadata, with the exception of orange that indicates cocci-shaped Sporosarcina strains. When exact GPS coordinates were not available, coordinates were approximated based on location data provided. The map was created using the Matplotlib and Basemap packages in Python, with rendering using GEOS (Geometry Engine - Open Source)

Fig. 2

Phylogenetic tree based on all the 16S rRNA gene sequences of the genus Sporosarcina deposited on the Ribosomal Database Project. Sequences were filtered to be greater or equal to 1200 bases long, good sequence quality, and both cultured and uncultured organisms. The sequences were aligned using SILVA, and refined using MUSCLE. The tree was built using FastTree and visualized in iTOL

Fig. 3

Average number of genes for 29 cocci Sporosarcina strains in the different categories of the clusters of orthologous groups (COG) using RPS-Blast against the National Center for Biotechnology Information conserved domain database (CDD). Error bars represent standard deviation

Fig. 4

The core and pan-genomes of cocci-shaped strains of Sporosarcina. Using BLASTp, and cutoff values of 90% amino acid identity across 90% of the gene, 221 conserved core genes were identified among the strains. The pan genome has 8610 genes using 30% identity across 70% of the gene as cutoff parameters

Fig. 5

Phylogenetic tree of the cocci-shaped Sporosarcina strains based on the core genome, and rooted based on the 16S rRNA gene tree. Tree was built using core genes shared at 90% amino acid sequence identity and 90% sequence coverage by all strains. Those sequences were concatenated in the same order for each genome, aligned using MAFFT, and the tree was built using FastTree. The genome names are colored to reflect the phenotypic class they were assigned during their initial isolation by Pregerson (1973). Spore genes found in B. subtilis and other spore-forming bacteria were protein-blasted to determine whether similar sequences exist in cocci-shaped strains of Sporosarcina

Fig. 6

Average amino acid identity matrix between the 29 cocci-shaped Sporosarcina strains sequenced during this study. Thick black boxes indicate species 95-96% cutoffs as proposed by Konstantinidis and Tiedje (2005), and were generated with the Genome-based distance matrix calculator website

Fig. 7

BLAST Atlases comparing the 29 cocci-shaped Sporosarcina genomes against one of two complete reference genomes (S204 and P33), circular plots were generated with CGView Comparison Tool using BLASTn. Genomes are arranged with the genetically closest to reference genome on the outer ring, and most distantly related on the inner most ring. a S204 reference genome; ordered from outer ring to inner ring: 1) Forward CDS, tRNA and rRNA; 2) Reverse CDS, tRNA and rRNA; 3) S204; 4) DSM 2281; 5) P19; 6) P12; 7) P10; 8) P29; 9) P31; 10) P30; 11) P32b; 12) P17a; 13) P20a; 14) P3; 15) P1; 16) P21c; 17) P16a; 18) P25; 19) P18a; 20) P2; 21) P8; 22) P16b; 23) P7; 24) P34; 25) P26b; 26) P32a; 27) P17b; 28) P37; 29) P35; 30) P33; 31) P13. b P33 reference genome; ordered from outer ring to inner ring: 1) Forward CDS, tRNA and rRNA; 2) Reverse CDS, tRNA and rRNA; 3) P33; 4) P37; 5) P35; 6) P12; 7) P10; 8) P17a; 9) P31; 10) P30; 11) P29; 12) P32b; 13) P19; 14) S204; 15) P3; 16) P17b; 17) P26b; 18) P20a; 19) P32a; 20) DSM 2281; 21) P34; 22) P21c; 23) P8; 24) P16a; 25) P25; 26) P1; 27) P16b; 28) P18a; 29) P7; 30) P2; 31) P13