Representative images are shown in Fig. 7. Despite increased expression in the tolC mutant of several fli, flh, mot, flg and fla genes, we observed
no difference between swimming motility of the tolC mutant and the wild-type strains, with both strains being able to swim (Fig. 7a). HM781-36B chemical structure Regarding swarming motility, we found that after 24 hours of incubation the tolC mutant displayed a higher surface motility than the wild-type strain (Fig. 7b), consistent with our gene expression data. The swarming behavior of wild-type and tolC mutant strains was markedly different from the expR + positive control strain Sm8530, which spread over the agar uniformly in all directions whilst the two first strains had a growth branching out from the center of the colony (Fig. 7b). S. meliloti cells stressed with acidic pH or increased osmotic pressure due
to salt or sucrose showed decreased expression of genes involved in chemotaxis and motility, consistent with the cell needing to conserve energy [30, 31, 33]. Why the tolC mutant has increased swarming motility is not known. Figure 7 Swimming (a) and swarming (b) tests. Swimming find more and swarming plates containing 0.3% and 0.6% purified agar, respectively, were spotted with 5 μl of late exponential S. meliloti cultures grown overnight in GMS medium. The photographs were taken after 1 day of incubation for swarming and 3 days for swimming at 30°C. Conclusions The transcriptomic data presented here indicate that the absence of functional TolC protein in S. meliloti compromises cell homeostasis as reflected by the concomitant increase in expression levels of many genes putatively involved in cytoplasmic and extracytoplasmic stress responses. Intracellular stress can possibly be caused by accumulation of proteins and metabolites that can not be secreted combined with oxidative stress. To ameliorate adverse effects, a RpoH-dependent response is triggered with an increase in Adenosine triphosphate the expression of many genes encoding products protecting
macromolecules like DNA, RNA and proteins and helping their turnover. Perturbations in the cell envelope caused by a potential accumulation of proteins such as the truncated TolC in the periplasm may have triggered a Cpx-dependent stress response with a set of genes encoding periplasmic proteases, chaperones and protein modifying enzymes having increased expression. Increased protein synthesis causes increased expression of the genes responsible for transcription, translation and energy producing pathways. The hypothetical higher metabolic demand was mirrored by increased expression of genes encoding nutrient uptake transport systems. Further support for our observations that cell envelope perturbation leads to extracytoplasmic and to oxidative stress comes from recent studies in Vibrio cholerae type II secretion mutants [24]. Sikora et al.