Similar to the L. sphaericus C3-41 genome [55], we did not find the homolog of rtp (replication terminator protein-encoding gene) in the chromosomal assembly of OT4b.31. A total of 42 hypothetical protein coding sequences were assigned as putative transposable elements, with ref 3 the most frequent families being IS66, IS110, IS1272 and IS3. In addition, five prophage regions were identified, of which one region is intact and 4 regions are incomplete. Lactobacillus phage C5 (intact), Bacillus phage 105, Clostridium phage c-st, Bacillus Phage SPP1 and Bacillus phage W�� predicted regions were allocated at contigs 34, 8, 15, 18 and 37, respectively. Only lysis proteins were predicted in phages C5 and c-st regions. The only genes remaining in the phage 105 region are those for coat proteins, integrase, and hypothetical and phage-like coding sequences.

This is probably the remnant of phage invasion and genome deterioration during evolution. In addition, any previously reported phages in the genome of L. sphaericus C3-41 are in the genome of OT4b.31. Two elements contain conserved domains from the Listeria pathogenicity island LIPI-1, functionally assigned as a thiol-activated cytolysin and a phosphatidylinositol phospholipase C. The first was confirmed to correspond to the L. sphaericus B354 sphaericolysin coding gene in contig 18 (H131_12483). Sphaericolysin B354 has been reported to be widespread across L. sphaericus DNA homology groups not only including IIA, IIB, IV and V [56] but also non-grouped species such as OT4b.31.

Upstream, in the same contig, a Bacillus toxin from the family Mtx2 (PFam PF03318) was found and described as a hypothetical Sip1A toxin coding sequence (H131_12498). Purified from Bacillus thuringiensis strain EG2158, Sip1A is a secreted insecticidal protein of 38 KDa having activity against Colorado Potato beetle (Leptinotarsa decemlineata) [57]. Considering that L. sphaericus OT4b.31 was isolated from beetle larvae, we suggest potential coleopteran larvicidal activity. To our knowledge, strain OT4b.31 is the first report of a predicted Sip1A-like toxin in a native Lysinibacillus sphaericus. Unexpectedly, mtx or bin mosquito pathogenic genes were not found in the OT4b.31 genome, despite a previous report showing positive evidence of BinA/B toxins with no larvicidal activity [10]. A total of 32 CDSs were described as surface (S) layer proteins or S-layer homologs (SLH).

Drug_discovery The putative S-layer gene sllB (H131_05299) previously reported in L. sphaericus JG-A12 [58] was found in a 3,696 bp sequence allocated in contig 8. Three sequences with conserved domains similar to Slp5 and Slp6 were identified in contigs 8 (H131_05339, H131_05344) and 22 (H131_16838). Bacillus sp. B-14905 was the most similar sequence for the majority of S-layer protein domains.