5 to 34.0 million years, which appear to be relatively similar to those values calculated for the 16S rRNA gene (Table 2). Luminescence in V. harveyi BB170 was induced when exposed to the supernatants of the amber bacteria tested. This was observed at 4 h in all the bacterial isolates tested,

which harbored luxS, SB431542 and was not the case for the negative control tested. Luminescence values are shown in Fig. 3, a (isolate 4_AG11AC10), b (isolate 10_AG11AC13a), and c (isolate 16_AG11AC14). The negative control (6_AG11AC11) did not emit statistically significant luminescence in any of the time points (Fig. 3d). Importantly, the luminescence emitted by the reporter strain in the presence of the putative AI_2 of all amber isolates tested is statistically significant, as shown by the one-way analysis of response (Fig. S1). The overlapping circles for each pair Student’s t and Best Hsu’s MCB also indicate significant difference between the three strains and the control. Our results are the first to report the presence and evolutionary rate of genes involved in QS in ancient bacteria. The amplification of luxS in several of the amber isolates tested is neither contamination

nor systematic errors of the PCRs. This was highly predicated by the luxS and 16S rRNA gene dendogram analyses, which clearly show a separation between the extant and ancient bacteria. Cross-contamination can also be discarded due to the differing high throughput screening assay 16S rRNA gene sequences among the isolates

that were positive for luxS. Moreover, all three sets of luxS primers were oxyclozanide tested in c. 130 amber isolates, regardless of being a Gram-positive or Gram-negative. If contamination of the primer sets would have occurred, luxS would have been amplified in all or most of the isolates tested. It should be noted that amber possesses preservative properties, representing an opportunity to isolate and extract suitable ancient DNA for analyses such as those performed in the present study (Cano, 1996). Most luxS sequences in the amber isolates were similar to the luxS sequences of extant Bacillus spp. when performing the blast search. This may be due to the unchanged nucleotide sequence of the amplified region of luxS. This may not have been the case for most of the Gram-negative bacteria tested (except for isolate 9_AG11AC12a), which were negative for luxS. This may suggest that Gram-negative bacteria lacked luxS millions of years ago or that these harbored luxS sequences different from those of present-day bacteria. The presence of a luxS sequence similar to that of Bacillus spp. in an ancient Gram-negative isolate (isolate 9_AG11AC12a) is a matter of further research as this could suggest the horizontal transmission of the gene between Gram-positive and Gram-negative bacteria. Cross-contamination is a possibility that can be discarded as this isolate was identified as a Brevundimonas sp. by a blast search of the 16S rRNA gene sequence.