These three genes were bordered by methyltransferase gene (mt1) at the upstream and putative orfC gene at downstream (Fig. 1a). A blast search indicates that OrfC contains a PAP2 domain

(type 2 phosphatidic acid phosphatase) and may be a PAP2-like superfamily member. In order to localize the promoter(s) for these three genes, RACE analyses were performed to determine the transcription start site(s). As shown in Fig. 1b, we were able to obtain a RACE fragment only from RACE-PCR reactions initiated within fimQ (lane 1), not from the reactions initiated within either high throughput screening assay fimP (lane 2) or srtC1 (lane 3). The size of the RACE fragment is consistent with the sequence-derived size of 590 bp. The results suggest that the transcription of either fimP or srtC1 was not initiated immediately upstream of these two respective genes. It is likely that both fimP and srtC1 were transcribed together with fimQ as a single mRNA unit. To confirm this, we amplified the junctional regions of fimQ-fimP and fimP-srtC1. As shown in Fig. 1c, lanes 1 and 3, when the same cDNA generated by the

use of primers 3 or 5 were used as the templates, both junctional regions were amplified. The two PCR products have the expected sizes of 540 and 712 bp. These results indicated that fimQ-fimP and fimP-srtC1 are together at the mRNA level. Therefore, these data confirmed that fimQ, fimP and srtC1 were transcribed as a single polycistronic mRNA. In addition, no amplification was observed when total RNA was used as the template find more (Fig. 1c, lanes 2 and 4). The results suggest that there is no DNA contamination in the RNA preparation and the amplicons produced were derived from the cDNA generated in the RACE reactions. When similar reverse transcriptase-PCR reactions were performed on the junctional regions of mt1-fimQ and srtC1-orfC, no amplicon was obtained (data not shown). These results reveal that fimQ

is the first gene and srtC1 is the last gene in a tricistronic operon. This assignment is consistent with our previous findings Methamphetamine that orfC is not required for the type 1 fimbriae biosynthesis (Chen et al., 2007). To locate the transcription start site, the amplified RACE PCR product (Fig. 1b, lane 1) was cloned into Zero Blunt TOPO vector and sequenced. Based on the DNA sequence obtained, the fimQ (and the fimP and srtC1) transcription start site was located at the adenine residue that is 58 bp upstream of the proposed ATG start codon (Fig. 1d). The identified transcription start point was subsequently used to deduce the putative promoter sequence of the type 1 fimbria gene cluster based on the consensus sequences observed in promoters from prokaryotic organisms. The deduced −35 (TTGGGG) and −10 (TACATT) boxes for the promoter of the gene cluster are separated by a spacer of 16 nucleotides (Fig. 1d).