Figure 3

Promoter activity, gene expression and primer extension analyses. A) Time course of promoter activity of pimT using the xylE gene (encoding catechol dioxygenase) as reporter. The enzyme activity of crude extracts was measured at the indicated culture times. Cells were grown in the presence (blue) or absence (red) of added homoserine (2 mg/ml). B) Gene expression analysis of pimT by RT-PCR. Analysis was carried out on S. natalensis wild type strain in the presence (+) or absence (-) of added homoserine or PI-factor as indicated in the Methods section after 28 (left panels) and 30 (right panels) PCR amplification cycles. The identity of each amplified product was corroborated by direct sequencing. The absence of contaminating DNA in the RNA samples was assessed by PCR. Transcription of the lysA gene (encoding diaminopimelate decarboxylase) located outside the pim cluster was also assessed as an internal control. The result of the analysis in the absence (-) or presence (+) of homoserine after 30 PCR cycles is shown. Identical result was observed in the presence of PI-factor. C) Mapping of the transcriptional start site of pimT by primer extension analysis. The full nucleotide sequence is shown in the upper fluorogram. The nucleotides corresponding to the end of the primer extension are highlighted. The arrow shows the direction of transcription. In the lower panel (D), the start of the transcript is indicated in bold. The -10 and -35 hexanucleotides are boxed, and the first codon is shown in boldface letters. Nucleotides showing homology with the 16S RNA that could form a ribosomal binding site are framed with a box labelled RBS.