coli and these potential modifications are a response to environmental stresses, specifically those associated with envelope MK-4827 molecular weight stress, such as pH, and this response is controlled by several regulatory pathways [46, 48]. We demonstrated that as the pH increases to 8.0, the Eagan isolate induced two gluconate permeases, one being part of an operon with gluconate metabolism genes, these likely providing the proteins and enzymes linked into energy production (through the

ED or PPP pathways) but also potentially providing other cellular alterations for coping with the stress (modifying the LOS, for instance). In contrast, the NTHi R3264 isolate did not induce the HI1010-1015 operon at pH 8.0. Consistent with this isolate inducing its biofilm formation at pH 8.0, it induced various, genetically unlinked iron acquisition genes (Table 3; the iron uptake genes hitAB, tbp1-tbp2 and hxuB were all upregulated and the iron storage ferritin gene was down-regulated). In multiple bacterial species iron acquisition pathways have been linked to the development of the biofilm lifestyle; such that if these pathways are removed or iron

is unavailable it depletes their biofilm-forming ability [16, 19]. Likewise in studies on NTHi biofilm formation and biofilm maturation, the iron uptake has been shown GDC-0941 concentration to be essential [17, 49–54]. It should be noted that in our comparative analyses of R3264 and Eagan at pH 8.0 we showed that Eagan did not form significant amounts of biofilm. As a comparison of their profile of growth pathways at pH 8.0 and then for R3264 at 6.8 (when R3264 cells forms less biofilm), the transcriptional switch in the planktonic R3264 cells at pH 8.0 compared to 6.8 is an indication of their response to this environmental condition and mechanisms that selleck kinase inhibitor predispose the cells to biofilm formation as well as allowing a direct comparison to the Eagan planktonic cells at pH 8.0. The R3264 cells at pH 8.0 that are in the biofilm were therefore excluded from our comparison; these

by definition would be greatly different (probably including the type IV pili or other adhesins) and not a clear comparison to the non-biofilm forming Eagan cells. It was not our aim to compare planktonic against biofilm cell but the response to increased Thymidylate synthase pH, conditions we know shift the R3264 cells to biofilm-forming state. It is worth noting that there were iron-associated genes up-regulated in Eagan at pH 8.0 but not to the extent observed in R3264. Table 3 Genes differentially expressed in H. influenzae R3264 at pH 8.0 compared to pH 6.8 Genes up-regulated at pH 8.0 compared to 6.8 Iron uptake genes Gene Log 2 fold p -value FDR Comment hitA 1.76 9.65×10-12 2.46×10-9 Iron uptake ABC, periplasmic domain hitB 1.31 8.77×10-7 1.11×10-4 Iron uptake ABC, permease domain tbp2 1.54 2.92×10-5 2.74×10-3 Iron-binding OM receptor tbp1 1.49 3.53×10-7 5.26×10-5 Transferrin binding protein h×uB 1.02 8.62×10-5 7.