## 3 A Brief Overview of AHL Systems in _Burkholderia_  
A comparison of _Burkholderia_ 16S rRNA sequences is known to yield three main taxonomical clusters/clades, the _B. cepacia_ complex (BCC), the _B. pseudomallei_ group (consisting of _B. mallei_ and _B. pseudomallei_) and the recently defined plant-beneficial (PBE) group [52]. Of the near 20 AHL signals described so far (mainly C4 to C18-homoserine lactones (HSL) as well as HSLs with the C3 position substituted or unsubstituted by an oxo, or hydroxyl group), _Burkholderia_ responds to six types of AHLs (Table 1). On the other hand, the topological arrangements of the AHL systems within the _Burkholderia_ genus show a broad variation. Briefly, _Burkholderia_ species contain one to three AHL systems that follow different topological arrangements, as summarized in Table 2.  
Out of the 16 local topological arrangements described in proteobacteria, 10 are found in _Burkholderia_ (comprising of completely sequenced genomes, draft genomes and individual Genbank entries), including a new topology type named here as M4 (\(\bar{R}\bar{M}X(<7)\bar{I}\) ), which had previously not been detected in Proteobacteria. Out of 35 completely sequenced _Burkholderia_ genomes, 32 (91%) were found to have AHL QS systems that belonged to eight distinct local topology types. In contrast, \begin{table}
\begin{tabular}{c c c} \hline \hline
**Symbol** & **Structure** & **Symbol** & **Structure** \\ \hline \(N\)-hexanoyl-\(L\)-Homoserine & & \(N\)-decanoyl-\(L\)-Homoserine lactone (**C10-HSL**) & \\ lactone(**C6-HSL**) & & \\ \hline \(N\)-octanoyl-\(L\)-Homoserine & & \(N\)-3-hydroxydecanoyl \\ lactone (**C8-HSL**) & & Homoserine lactone (**OHC10-HSL**) & \\ \hline \(N\)-3-hydroxy-octanoyl & & \(N\)-3-oxo-tetradecanoyl-\(L\)-homoserine lactone (**OC14-HSL**) & \\ Homoserine lactone & & \\ lactone & & \\ \hline \((\)**OHC8-HSL**) & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Chemical structure of \(N\)-acylated homoserine lactone (AHL) signals used by LuxR in the known species of the genus _Burkholderia_. out of 44 completely sequenced genomes of _Pseudomonads_, only 11 (25%) had AHL QS genes that belonged to three topology types. Two of the three topology types, R1 ( \(\bar{R}\bar{I}\) ) and L1 ( \(\bar{R}\bar{L}\bar{I}\) ) were also present in _Burkholderia_. While the arrangements of AHL genes of pseudomonads are apparently simpler and have less intervening sequences between the R and I genes, the _Burkholderia_ genus, especially the _B. pseudomallei_ group, has a number of more complex arrangements. The cladograms of LuxR and LuxI proteins show a seemingly complex classification scheme which can however be explained by the local topology of their AHL systems (Supplementary material). Namely, the I and R genes seem to classify according to their local topology, rather than according to the species. For instance, an I gene with a given topology within a species (e.g., \(\bar{R}\bar{I}\) in _B. pseudomallei_ 1710b) is consistently more similar to an I gene of identical topology within another species ( \(\bar{R}\bar{I}\) in _B.thailandensis_ E264), than to an I gene of the same species, but having a different local topology (e.g., \(\bar{R}\bar{M}\bar{I}\) in _B. pseudomallei_ ). It thus appears that local topology within the chromosome may have evolved before the separation of various _Burkholderia_ clades. As schematically shown in Table 3 and Figure 1, the chemical structure of the AHL signals is in good correlation with the local topology of the AHL systems, _i.e._, the same chemical signal is produced by AHL systems belonging to identical or related local topologies, or to a few topology types. The agreement across genera is less equivocal (details not shown). For instance, an AHL system of L1 topology in _B. xenovorans_ is regulated by the signal OC14, while an analogous AHL system in _P. aeruginosa_ is regulated by a different signal, OC12.