## 3 Results and Discussion
### Biosynthetic Genes

Table 2: List of potential methylketone homologs and their distribution in clusters, as calculated by the Sequence Similarity Network (SSN). The first biggest cluster was composed by proteins from different species of _Bacillus_, including _B. nematocida_ (Table 2), indicating that the proteins that are mostly related with putative methylketone synthase used as starting point in this study are found within this genus. The second cluster composed exclusively by _Gilliamella apicola_ indicates that this species, widely distributed in the honey bee environment [23; 24], is a potential producer of 2-heptanone. The compositions of the other minor clusters suggests that putative methylketone synthase homologs may be found in Acetobacteraceae, as well as in _Bartonella apis_, _Parasaccharibacter apium_ and _Bombella apis_, well-known symbionts of honey bees [23; 25; 38; 39], and, among Lactobacillusae, in _Pediococcus acidilactici_, recently shown to have a protective effect towards honey bees [40]. Most of the bacterial thioesterases described in Table 2 have yet to be functionally characterized. The role of these enzymes has been studied more extensively in plants, where acyl thioesterases are reported to produce fatty acids that play roles in plant-inset and plant-microbial interactions [41]. A similar role of these enzymes in bacteria-bacteria and bacteria-insect interactions may be hypothesized and be the object of further studies. The overall mean distance of the sequences listed in Table 2 was 1.33 \(\pm\) 0.07, and the pairwise distance of the representative sequences from each cluster highlighted that the most diverse were those of _B. apis_ from cluster 7 and _B. cereus_ from cluster 4 (Figure 4). \begin{table}
\begin{tabular}{l l c c} \hline
**Cluster** & & **UniProt ID** & **Description** & **Species** \\
**N.** & & & & _Bacillus thuringiensis_ subsp. \\ \hline
5 & A0A1L2Z0L8 & 114-dihydroxy-2-naphthoyl-CoA hydrolase & _Bacillus thuringiensis_ subsp. \\
5 & A0A2A2 NYU0 & Thioesterase & _Bacillus tyopensis_ \\
5 & A0A2A7 HHQ3 & Acyl-CoA thioesterase & _Bacillus cereus_ \\
5 & A0A2A7W716 & Thioesterase & _Bacillus wiedmannni_ \\
5 & A0A2B8 HQR2 & Acyl-CoA thioesterase & _Bacillus thuringiensis_ \\
5 & A0A4L2U5Z0 & Acyl-CoA thioesterase & _Bacillus cereus_ \\ \hline
6 & A0A1B9 JQR5 & Tol-pal system-associated acyl-CoA thioesterase & _Gilliamella apicola_ \\
6 & A0A1B9M1U5 & Tol-pal system-associated acyl-CoA thioesterase & _Gilliamella apicola_ \\
6 & A0A1B9 MHC5 & Tol-pal system-associated acyl-CoA thioesterase & _Gilliamella apicola_ \\
6 & A0A1B9 Nl25 & Tol-pal system-associated acyl-CoA thioesterase & _Gilliamella apicola_ \\ \hline
7 & A0A1R0 FB95 & (3S)-malyl-CoA thioesterase & _Bartonella apis_ \\
7 & A0A1U9 ME42 & (3S)-malyl-CoA thioesterase & _Bartonella apis_ \\
7 & A0A1U9 MKW6 & (3S)-malyl-CoA thioesterase & _Bartonella apis_ \\ \hline
8 & A0A2C6J477 & 4-hydroxybenzoylzyl-CoA thioesterase & _Parasaccharibacter apium_ \\
8 & A0A6N7F3K1 & YbgC/FadM family acyl-CoA thioesterase & _Bombella apis_ \\
8 & A0A7U7C6Z5 & 4-hydroxybenzoylzyl-CoA thioesterase family active site & _Parasaccharibacter apium_ \\ \hline
9 & A0A062X6R3 & Acyl-CoA thioester hydrolase \textbackslash{}^{}\text{}\text{}\text{}\text{}\text{}\text{}\text{} \text{}\text{}\text{}\text{}\text{}\text{}\text{}\text{}\text{}\text{} \text{}\text{}\text{}\text{}\text{}\text{}\text{}\text{}\text{}  
GNDs representing genomic regions around the genes encoded for the sequences from the submitted SSN highlighted the presence of two main protein families in the analyzed sequences: the 4 HBT Thioesterase superfamily (PF03061), shared by clusters 1 (_Bacillus_), 3 (Acetobacteraceae), 6 (_Gilliamella_), 7 (_Bartonella_) and 8 (Acetobacteraceae) (Figure 5), and the 4 HBT_2 Thioesterase-like superfamily (PF13279), shared by clusters 2 (_Gilliamella_), 4 (_Bacillus_) and 5 (_Bacillus_). The Acyl-ACP thioesterase (PF01643) protein family was found in Lactobacillus from cluster 9, suggesting that this family may have evolved separately.