Nevertheless, they observed the induction of an RpoE-mediated stress response, whilst we observed a Cpx-mediated stress response, emphasising the differences between the two types of mutations/organisms. Responses to stress caused by S. meliloti lack of functional TolC are distinct from other stress conditions such as osmotic
shock and acid pH [30, 33]. In the latter two there is general shut-down of the expression of genes involved in central metabolism, protein metabolism, iron uptake and chemotaxis. In contrast, the tolC mutant shows an increased expression of genes involved in all of these pathways. One possible explanation could be the higher need for energy and reducing LB-100 supplier NU7026 purchase power to combat oxidative stress and the possible accumulation of proteins that can not be secreted. Another possibility is related to an eventually compromised electrochemical proton gradient across the membrane. Since TolC is the outer membrane component of many transport systems
, its inactivation may affect both proton transport and ATP synthesis and possibly the cell responds by increasing expression of genes involved in central metabolism to synthesize more ATP. Although many questions remain unanswered, our results highlight the mechanisms by which a large number of genes act together to restore cell homeostasis and, in particular, points to TolC protein as being fundamental in the biology of this microorganism. Methods Bacterial strains and growth conditions Bacterial strains used in this study were wild-type S. meliloti 1021 (Sm1021) , SmLM030-2 (Sm1021, pLS378 integrated into the tolC gene region) , Sm8530
(Sm1021, expR +) , and Rem::Tn-5 (Sm1021, rem -) . For gene expression profiling, overnight cultures of S. meliloti 1021 and tolC mutant strain SmLM030-2 grown in TY complex medium  were diluted Roflumilast to an initial OD600 = 0.1 in GMS medium (Zevenhuizen, 1986). Triplicate flasks of each strain were cultured at 30°C in GMS medium at 180 rpm for 20 hours. Isolation and processing of RNA samples Cells were harvested, resuspended in RNAprotect bacteria reagent (see more Qiagen), and total RNA extraction was carried out using the RNeasy MiniKit (Qiagen) with DNase treatment following manufacturer’s recommendations. Once absence of residual DNA was confirmed, concentration and purity were determined using a Nanodrop ND-1000 UV-visible spectrophotometer. RNA integrity was checked with an Agilent 2100 Bioanalyser using a RNA Nano assay (Agilent Technologies). RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Medicago/Sinorhizobium Genome Arrays, according to the manufacturer’s Prokaryotic Target Preparation Assay.