Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 Page 9 of 17 Figure 4 Identification of mutants with increased chitin content. Depicted histograms correspond to flow cytometry analysis of yeast cells after treatment with the chitin-binding fluorescent dye, Calcofluor white (5 μg/ml for 10 min). Representative experiments of mutants are shown in which chitin content was found to be higher compared to the wild-type strain. For each mutant (blue line), a histogram corresponding to the wild-type strain (filled area) handled in parallel is included in each representation. The name of genes functionally related to cell wall integrity and morphogenesis is underlined. “RM” refers to the ratio of means of fluorescence intensity for each cell population (mutant/wt). levels of Slt2 activation, were transformed with a repor- ter construction where the promoter region of the MLP1 gene was fused to the lacZ coding sequence (MLP1P-lacZ) and transcriptional activation was studied under standard growth conditions by measuring b-Galactosidase activity. As shown in Figure 6a, a good correlation between the expression levels of MLP1 and Slt2 phosphorylation (Pearson’s correlation coefficient of 0.8) was observed except for the mutant lea1Δ, in which the reporter expression was significantly lower than expected from the MAPK phosphorylation status. Simi- lar results were obtained when using the CWP1 promo- ter, another reporter of the CWI pathway (Figure 6b). In this regard, comparable behavior was recently described for the mutant msg5Δ in which Slt2 phosphorylation is not associated with Rlm1-dependent transcription [78]. On the basis of this observation, the existence of addi- tional S. cerevisiae mutants with the same behavior cannot be ruled out. In contrast to the group of mutants described above with increased levels of phospho-Slt2, another group of 110 mutants selected in the screening did not have detectable differences in Slt2 phosphorylation with respect to the wild-type strain. In order to confirm the nourseothricin resistance of these strains, we trans- formed them all with the reporter construct (MLP1P- NAT1) and carried out minimal inhibitory concentration (MIC) assays using a microdilution method. Eventually, 38 mutant strains recorded higher antibiotic MIC values than that of the wild-type (Additional file 2), confirming the phenotype of nourseothricin resistance, whereas all the other mutants behaved the same as the wild-type