Reactions with no addition of reverse transcriptase served as negative control and proved the absence of DNA contamination. Specificity of amplification was assessed by analyzing the melting curve of the amplification product. Primers to amplify lscB were used MRT67307 mouse for constructs lscB and lscA Up B while primers to amplify lscA were used for constructs lscA, lscB UpN A and lscB Up A. All the results were normalized to amplification of the cDNA of gyrA (PSPPH3667) as described previously . Analysis of lscA gene expression by
Reverse-Transcriptase polymerase chain reaction (RT-PCR) Template-specific primers were designed for the respective lscA variants of P. syringae pv. selleck inhibitor glycinea PG4180, pv. phaseolicola 1448A, pv. syringae B728a, and pv. tomato DC3000. Bacterial cells were grown in HSC
medium and harvested at an OD600 of 0.5 as well as 2.0. RNA was extracted by acid phenol/chloroform extraction method . An RT-PCR was performed on total mRNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas) as recommended by the manufacturer. The strain-specific lscA primers were used to check for presence of an lscA mRNA by PCR using cDNA as template. Regular PCR with the same primer-pairs and genomic DNA as template were used as controls. The thermocycler program was as follows: 1 cycle of 95°C for 90 s; 25 cycles of 95°C for 15 s, 66°C for 15 s, 72°C for 30 s; 1 cycle of 72°C for 5 min. The results were analyzed by 1% agarose gel electrophoresis. Bioinformatics analyses Vector NTI Advance 10.1.1 (Life Technologies, California, USA) was used for the nucleotide, amino acid sequence alignments, as well as for generating genetic maps. BLAST-N and BLAST-P programs were used for online sequence analyses . The website http://www.pseudomonas.com was consulted for the determination of P. syringae gene orthologs and paralogs . Authors’ information SK – Department of Molecular Microbiology, Molecular Life Sciences Research Center, Jacobs University Bremen,
Germany; ASr – Current Address: Department of Experimental Limnology, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Epothilone B (EPO906, Patupilone) Stechlin, Germany; DP – Department of Biochemical Engineering, Molecular Life Sciences Research Center, Jacobs University Bremen, Germany; ASt – Department of Molecular Microbiology, Molecular Life Sciences Research Center, Jacobs University Bremen, Germany; MU – Department of Molecular Microbiology, Molecular Life Sciences Research Center, Jacobs University Bremen, Germany. Acknowledgements We thank Helge Weingart for his helpful comments and Ramesh Mavathur for his help with Sanger sequencing. This study was supported by the Deutsche Forschungsgemeinschaft (UL-169/5-1). References 1.