For each strain pili of at least six bacteria were counted;

error bars indicate deviations from mean values. Strain-specific expression of pili subunits To analyze the molecular basis of strain-specific differences in pili formation, RNA hybridization PD173074 mouse experiments were carried out to study the mRNA levels of the C. diphtheriae spa genes. These genes are organized in three different clusters together with the corresponding sortase-encoding genes in the sequenced strain NCTC13129 [13, 19]. The first cluster comprises the genes spaA, spaB, and spaC, which are most likely organized as an operon; the second cluster is formed by spaD and a putative spaE-spaF operon, and a third cluster comprises the spaG, spaH, and spaI gene, which are most likely independently transcribed. Strain-specific differences were detected, when probes for the detection of all genes of cluster I and III were applied in RNA hybridization experiments (Fig. 6A). Figure 6 Strain-specific distribution and expression of pili-encoding genes. (A) Levels of spa gene transcripts

in different C. diphtheriae strains. Total RNA was isolated from the indicated C. diphtheriae strains and hybridized with probes monitoring 16SrRNA for control as well as spa gene transcription. (B) PCR detection of spa genes. Chromosomal DNA of the indicated C. diphtheriae strains was used as template for PCR using specific oligonucleotide pairs Dorsomorphin solubility dmso for the spa genes indicated at the right side of the figure. Strongest hybridization signals with spaA, spaB, and spaC probes were detected with RNA isolated from strains ISS4746 and ISS4749, slightly lower signal intensities were observed with strain DSM43989, while only faint signals were obtained for cluster I

mRNA for the other investigated Thymidylate synthase strains. Strong transcription of spaG, spaH, and spaI were again detected in strains ISS4746 and ISS4749, while other strains did not express cluster III genes deduced from RNA hybridization experiments. The data are in accordance with the AFM experiments presented in Fig. 5, which show formation of a high number of extended pili for strains ISS4746 and ISS4749, followed by DSM43989; however, hybridization signals may differ not only due to mRNA abundance, but also due to sequence alteration. To elucidate whether the missing transcripts in various strains are the result of regulatory processes or have genetic reasons, PCR experiments were carried out, which showed that missing transcripts are correlated to lacking PCR products making regulatory effects unlikely (Fig. 6B). Furthermore, reproducible strain-specific differences in sizes of the PCR products were observed for spaA and in band intensities for spaB fragments, Selleck G418 suggesting that also sequence deviations exist besides strain-specific differences in the spa gene repertoire.