RNA isolation, RT-PCR, and 5′ RACE.

Total cellular RNA was isolated from S. sanguinis according to a method described previously by Chen et al. (37) and further purified using an RNeasy purification kit (Qiagen). To determine whether a contiguous transcript exists between the neighboring genes by RT-PCR, cDNA was generated from 2 µg of purified RNA using avian myeloblastosis virus (AMV) reverse transcriptase and random primers (Promega). Five percent of the generated cDNA was then used in a PCR. The transcriptional start site(s) of the pil cluster was determined using the 5′ RACE system (Invitrogen). Five micrograms of total cellular RNA from S. sanguinis SK36 was used in a RACE reaction. cDNA was generated from total RNA using three SSA_2318-specific primers, pil19960S, pil19730S, and pil19810S, which contain the antisense sequence of SSA_2318 and are located 226, 456, and 376 bases 3′ to the translation start site of SSA_2318, respectively. RNA was isolated from three independent cultures, and samples from each culture were used in the RACE analysis. After the addition of the poly(C) tail to the cDNA pieces, the abridged anchor primer (AAP) was paired with primers pil20050S, pil19845S, and pil19890S to amplify the pil-specific cDNA. A total of 0.1% of the resulting PCR product was reamplified by nested PCR using primer pil20180S and the abridged universal amplification primer (AUAP). The final PCR products were separated by gel electrophoresis and purified individually for sequencing analysis.