marcescens BMN 673 mouse strain 12 (67% identity), SmaI (CAB92553) from Serratia strain ATCC 39006 (60% identity). The AHL synthases SwrI and SmaI catalyze preferentially the synthesis of C4-HSL and, in less amount, C6-HSL [16, 37, 38]. To examine the evolutionary relationship between the LuxI family members described above, a phylogenetic analysis was performed using MEGA 4 and the neighbour-joining tree was showed in Figure 1. The results were consistent with the similarity analysis of amino acid sequences within LuxI family members, the LuxI family synthases were clustered into two groups, and SplI and SpsI from strain G3 are classified into group A and group B, respectively. Figure
1 Neighbour-joining tree of LuxI family members in Serratia. The phylogenetic tree was see more generated using MEGA 4. LuxI family members in Serratia are clustered into two groups according to the AHL patterns. SplI and SpsI from G3 were in group A and group B, respectively. The significance of each branch is bootstrap value calculated for 1000 subsets. Scale bar indicates the mean number of substitutions per site. SplI and SpsI from S. plymuthica G3 produce multiple AHLs To determine which AHLs were made by each SplI and SpsI, LC-MS/MS analysis was performed on extracted culture supernatants from the wild type G3 strain click here as well as recombinant E. coli strains expressing splI or spsI and the spectra
profiles compared to that of synthetic AHL standards. At least ten different AHLs were detected in varying abundance in the wild type G3, including unsubstituted AHLs (C4-HSL, C5-HSL, C6-HSL, C7-HSL, C8-HSL), 3-oxo derivatives (3-oxo-C6-HSL, 3-oxo-C7-HSL, 3-oxo-C8-HSL) and 3-hydroxy derivatives (3-hydroxy-C6-HSL, 3-hydroxy-C8-HSL). The most abundant and hence most likely biologically relevant AHLs detected in the spent culture supernatants of the endophytic strain G3 were 3-oxo-C6-HSL, C4-HSL, C6-HSL, 3-hydroxy-C6-HSL and 3-oxo-C7-HSL. However, strain G3 did not produce long chain AHLs . When expressed in E. coli (Table 2),
the recombinant SplI produced all ten Mirabegron AHLs whereas SpsI produced only unsubstituted AHLs, including C4-HSL, C5-HSL, C6-HSL, C7-HSL, and C8-HSL. The most abundant one was C4-HSL from SpsI, 100 fold higher than that the production of this molecule by SplI in E. coli, suggesting that SpsI is could also be the main AHL synthase responsible for synthesis of this AHL in G3, in accordance with SwrI and SmaI from different S. marcescens strains [37, 38] which share similarity to SpsI. Both SpsI and SplI produce C6-HSL, but only SplI was responsible for the most abundant signal 3-oxo-C6-HSL, that is similar to SplI from S. plymuthica strains HRO-C48 and RVH1 [14, 32], SprI from S. proteamaculans B5a, SpnI from S. marcescens SS-1 [34, 35], as well as EsaI from P. stewartii .