Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 ability to partner a variety of other transcriptional regula- tors under different conditions. It has previously been shown that Skn7 may be activated by Rho1p in response to cell wall stress [59], whereas Mog1p is a protein involved in nucleocytoplasmic transport. At the same time, Mog1 is required for optimal recruitment of Skn7 to specific gene promoters [60]. The fact that Slt2 is hyperactivated in these mutants suggests novel connec- tions between the CWI pathway and the cellular pro- cesses controlled by these elements. Regarding the set of mutants related to transcription, many of the proteins identified are involved in RNA poly- merase II dependent transcription controlling responses to a variety of conditions such as, heat stress (RTR1), oxi- dative stress (YPR115w) and anaerobic conditions (ROX1). Additionally, as visualized in the network map (Figure 3), there is a connection between the transcrip- tion factors Skn7 and Rox1, with both participating in the transcriptional response to oxidative stress [61]. Also, we identified two components of a module of the media- tor complex (Ssn3 and Ssn8), involved in the regulation of Skn7 activity [62]. The appearance of these mutants in our study suggests a functional link between them and the CWI pathway. These insights enable an association to be made between this MAPK pathway and additional stressful cellular events. In this regard, it is also worth to mention that some of the mutants identified in our screening have previously shown altered sensitivity to osmotic stress (phm8Δ, glg2Δ and mrs3Δ) or heat stress (ncl1Δ, suv3Δ and vps64Δ). A connection has been described between the CWI pathway and endoplasmic reticulum (ER) stress. When a cell encounters conditions that increase misfolded pro- teins, the Unfolded Protein Response (UPR) is activated to compensate for high levels of ER stress [63]. The Slt2 MAPK pathway is activated during ER stress [23], while UPR is activated by signaling through the CWI pathway during cell wall stress [64]. Moreover, a second pathway, the ER stress surveillance pathway (ERSU) independent of the UPR, has recently been linked to Slt2 activation [65]. According to our results, in some of the selected mutants these mechanisms of Slt2 activation could be involved. In fact, Emp47 is required for the export of spe- cific glycoprotein cargo from the endoplasmic reticulum, Sec72 is involved in targeting secretory proteins to ER, and Orm2 is a protein related to lipid homeostasis and protein quality control, being required for resistance to agents that induce UPR [66]. In addition, Orm2 interacts with Slt2, but the biological significance of this interac- tion is still unknown [67]. Finally, we found the cne1 mutant to be associated with ER quality control mechan- isms. Cne1 is a calnexin homologue of Saccharomyces cerevisiae that may play a part in the degradation of mis- folded glycoproteins. Page 7 of 17 The identification of deletant strains in genes whose function remains uncharacterized and those not pre- viously associated to cell wall integrity, both recording an increase in Slt2 phosphorylation, was of special interest since they could be putatively associated with cell wall construction or regulation. To further investigate this possibility, a phenotypical analysis was conducted on the 11 deletant strains corresponding to genes of unknown function and 15 without clear cell wall phenotypes reported in yeast databases. Thus, the sensitivity to Congo red, caspofungin, hygromycin B, caffeine and SDS was determined. These compounds affect cell integrity through different modes of action, whereby the dye Congo red interferes with proper cell wall assembly [68], caspofungin consists of a b-1,3-glucan synthase inhibitor, hygromycin B hypersensitivity has been associated with glycosylation defects [69], SDS is a detergent that affects membrane stability and also, indirectly, cell wall con- struction (increased accessibility) [70], and caffeine is a substance that indirectly activates the CWI pathway in a TOR1-dependent fashion [71]. Eventually, 15 out of 26 strains analyzed displayed altered sensitivity in at least one of the tests described (Table 2), suggesting that the activation of the CWI pathway in these mutants could be due to direct or indirect cell wall alterations. Interest- ingly, these mutants generally had more than one pheno- type supporting the existence of relevant cell wall damage. In contrast, identification of mutants without apparent cell wall defects could be related to the possibi- lity of CWI pathway activation by other stimuli. In this regard, the coordination under specific growth conditions between the CWI and other regulatory MAPK pathways has been extensively reported [20,25]. Further supporting this, Harrison and colleagues [42] suggested that the acti- vation of the CWI pathway by different stresses, rather than operating in a linear “top-down” manner, would provide lateral inputs that impact this regulatory pathway at different levels. Moreover, recent findings connect Slt2 MAPK to DNA damage responses [72]. Differences in chitin content between mutants with Slt2 activation The yeast cell wall normally contains approximately 2% chitin. However, certain mutations affecting cell wall stability increase chitin levels to as much as 20% of total wall polymers [73]. As this emergency response for cell wall repair is dependent on CWI pathway signaling, it prompted us to assess this response in the whole group of 64 mutants with basal activation of the pathway. Chitin content was measured by means of flow cytome- try after staining the cells with the chitin-binding dye, Calcofluor white (CW). This is a reliable and sensitive method for chitin determination, since it has been established a linear relationship between fluorescence