Molecular clocks for luxS were estimated similarly. To evaluate the expression of luxS in the amber isolates, luminescence assays were performed using isolates 4_AG11AC10, 10_AG11AC13a, and 16_AG11AC14 and V. harveyi BB170 as the reporter strain. Amber isolate 6_AG11AC11 was used as negative control as it lacked luxS. The criteria for selection of the isolates for the assays included differences between the amplified region of the 16S rRNA gene and cell morphology. For these experiments, the growth curves of the amber isolates were determined by OD600 nm measurements of aliquots collected (in triplicate) every 2 h for up to 8 h. Aliquots were filtered and added to a final concentration of 10% to the reporter strain

(final OD600 nm = 0.1). Luminescence emitted by the reporter strain in the presence of the putative

AI-2 was measured using a luminometer and is reported as relative light units (RLU). Background luminescence or find more the luminescence emitted by the reporter strain in the absence of bacterial filtrates was measured as well. Results are reported as plots of the luminescence emitted by the reporter strain in the presence of the supernatant of the amber isolates, and OD600 nm measurements are selleck screening library shown as well (y-axis). The x-axis represents the timing of the response of V. harveyi BB170 after addition of the putative AI-2. Sequence data matrices were log-transformed, and similarity matrices were used to construct dendograms using Primer E, version 6 (Clarke & Gorley, 2006). For the luminescence data, one-way analysis was performed to test for differences between group means using jmp pro 10 statistical analysis software (Statistical Discovery™, SAS Institute, Inc.). A total of 20 amber isolates were included in the present study Pyruvate dehydrogenase (Table S3). luxS was not amplified in most of the Gram-negative isolates, with the exception of isolate 9_AG11AC12a. The tree topology of luxS in the present study is comparable to that reported previously (Lerat & Moran, 2004). The amplified region of luxS clustered more closely to the luxS of B. megaterium (Fig. 1a).

This was not the case, however, for the 16S rRNA gene phylogeny, where several amber isolates formed distinct branches and clustered with differing bacteria genera (Fig. 1b). The dendogram of the luxS clearly showed separate clusters for the extant and ancient taxa (Fig. 2a), while the dendrogram of the 16S rRNA gene sequences showed a similar clustering of the samples by age (extant vs. ancient) (Fig. 2b). The evolutionary rate or molecular clocks for luxS and 16S rRNA gene sequences were calculated. The criteria for selection of the isolates included identification at the species level by blast searches of the 16S rRNA gene partial sequences. The evolution rate of the 16S rRNA gene of the amber isolates tested is shown in Table 1 and was estimated to be 14.5–30.3 million years. The results are consistent with the estimated age of the isolates (Table S1).