The genes of the che operon as well as the fla genes CE, F, G, H, I, J are cotranscribed [43, 55], so not all genes needed to be analyzed separately. cheR and cheY were chosen for analysis because cheR is at the border of the che operon, next to the deleted genes, and cheY was an additional control. cheC2 and cheW2, which

are not located in the che operon, were not tested separately, because the deletion of these genes does not cause a smooth-swimming phenotype (unpublished observations). Additionally, flaH was chosen as a representative of the fla genes, although a defect in Fla protein expression seemed a priori unlikely since no motility defect was observed. Table Dabrafenib chemical structure 2 Che and Fla protein expression in deletion strains. Strain Clone CheR CheY FlaH Δ1 1 1.24 1.06 1.76   2 1.11 1.21 1.28 Δ2 1 1.58 -1.46 1.39   2 1.00 -1.37 1.30 Δ4 1 1.24 1.08 2.03   2 -1.05 1.46 1.65 Δ2–4 1 1.14 -1.16 1.77   2 -1.96 -1.45 -1.37 The mRNA levels in the deletions in S9 were determined by qRT-PCR. Given is the fold difference in the mRNA level of the deletions compared to S9 Deforolimus chemical structure wildtype. Each result is the average of two replicates.

The qRT-PCR curves were analyzed using the method with normalization to the constitutively expressed fdx gene [56]. In none of the tested cases was a significant difference between deletion and wildtype observed. Complementation of the deletion strains reverted their phenotype to that of wildtype All deletions in the S9 background were complemented by reintroducing the deleted gene in cis. The phenotype of the complementations was examined by swarm plates and, for the single deletions, by motion analysis. In these assays, all complementations behaved exactly like the wild-type strain (see Additional file 5), confirming that the phenotypes observed in the mutants were a direct result of their gene

deletions. Bioinformatics analysis Tolmetin To collect information on the three unknown proteins and to test if the findings obtained in H. salinarum are potentially transferable to other archaeal species, a bioinformatics analysis was done. The starting point was a homology search and querying databases like COG [57] and Pfam [58]. The goal was to identify orthologs from other organisms for which some knowledge might exist, and to unravel correlations between the occurrence of the here investigated proteins and Che and Fla proteins. For this, an extensive search for Che and Fla orthologs in all published archaeal genomes was performed (see Additional file 6). OE2401F is classified as a HEAT_PBS or HEAT family protein [58]. These proteins are predicted to contain short bi-helical repeats. Beside the HEAT-like repeats, no other domain could be detected.