Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 Page 10 of 17 Figure 5 Slt2 activation in gat2Δ and lea1Δ mutants is not transmitted via Rom2 nor related to variations in chitin content. (a) Analysis of chitin content of gat2Δ and lea1Δ cells after Calcofluor white staining by means of flow cytometry as described in Figure 4. (b) Western blots detecting Phospho-Slt2 (P-Slt2) and actin as loading control in total protein extracts from single (gat2Δ and lea1Δ) and double (lea1Δrom2Δ, gat2Δrom2Δ, lea1Δbck1Δ and gat2Δbck1Δ) mutant strains. strain. Bearing in mind that this phenotype was also confirmed for the 64 mutants with increased phosphory- lation of Slt2, this group probably includes mutants with antibiotic resistance by non-CWI-related mechanisms, such as alternative effects on MLP1 expression or intrin- sic antibiotic resistance. To further investigate the molecular mechanism involved in the group of antibiotic resistant strains with- out detectable variation in phospho-Slt2 levels, the MLP1P-lacZ reporter was used to monitor levels of expression of MLP1 in this set of mutants. For the major- ity of the mutants, MLP1 expression levels were low and in general higher than the wild-type strain (Figure 7a). However, mutant strains ssd1Δ and pmt2Δ had very high levels of MLP1 expression (Figure 7a) in spite of very slight, if any, Slt2 activation (Figure 7b). Pmt2 catalyzes the first step in O-mannosylation of target proteins [79] and SSD1 has been linked to cell wall integrity [80,81]. To elucidate a possible participation of the CWI pathway in the activation of the gene expression in these mutants, we proceeded to generate pmt2Δslt2Δ, pmt2Δrlm1Δ, ssd1Δslt2Δ and ssd1Δrlm1Δ double mutants. By using these strains transformed with plasmids containing tran- scriptional fusions of MLP1, CWP1 and SED1 to lacZ, we were able to delimit the requirement of the CWI MAPK and its main transcription factor (Rlm1) for the observed transcriptional up-regulation. As shown in Figure 8a, gene activation in the absence of both CWI pathway ele- ments, Slt2 or Rlm1, was completely annulled compared to pmt2Δ and ssd1Δ single mutants. These results point to the existence of situations where undetectable changes in MAPK activation (Figure 7b) give rise to remarkable consequences at gene expression levels. As further proof of the essentiality of Slt2 activity in the transcriptional