Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 cell wall integrity, particularly those of unknown function, as well as genes related to transcription, RNA and protein metabolism and transport. Although an increase in chitin content has been described as one of the effector mechanisms within the compensatory response, our results show that there is not always a correlation between activation of the CWI pathway and chitin levels. This effect is probably depen- dent on the stimuli involved. In fact, within the group of mutants with higher chitin levels, the functional group of genes related to cell wall and morphogenesis is overre- presented, suggesting a functional link between the acti- vation of the chitin deposition-mediated mechanism and cell wall defects. Another aspect of interest is the lack of uniformity of the magnitude of MAPK activation and the transcriptional out- puts in the mutants analyzed, suggesting that their modula- tion could be relevant for cellular adaptation to specific circumstances. Although levels of MAPK activation gener- ally correlate with transcriptional up-regulation, there are also, and unexpectedly, mutants with a transcriptional acti- vation dependent on a functional Slt2 MAPK and the tran- scription factor Rlm1, despite not having significant levels of phospho-Slt2. Bearing in mind that this phenomenon could take place in other circumstances, MAPK pathway related studies should seek information on both MAPK activation and gene expression to discover whether the pathway plays a role under specific conditions. Methods Strains and media Experiments were performed with the full collection of Saccharomyces cerevisiae strains (BY4741 background, MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) individually deleted in all of the ORFs identified in this organism (4840) that were replaced by the Geneticin resistance- codifying KanMX4 module. This collection was provided by Euroscarf (Germany). Double mutants slt2Δpmt2Δ, slt2Δssd1Δ, rlm1Δpmt2Δ, rlm1Δssd1Δ, gat2Δrom2Δ, lea1Δrom2Δ, gat2Δbck1Δ and lea1Δbck1Δ were primar- ily obtained by directly replacing the genes SLT2 or RLM1 and ROM2 or BCK1 in the corresponding pmt2Δ or ssd1Δ and gat2Δ or lea1Δ single-deletant-strains, respectively, with the HIS3 marker using the SFH PCR- based method described by Wach and colleagues [82]. Alternatively, slt2Δ::HIS3, rlm1Δ::HIS3, rom2Δ::HIS3 or bck1Δ::HIS3 strains constructed in the wild-type BY4742 background (MATa) were crossed with the single dele- tants of interest to generate the corresponding heterozy- gous disruptants. After sporulation and tetrad analyses of these strains with standard yeast genetic techniques, hap- loid double-mutant segregants were selected. Correct ORF replacements were verified by PCR. Page 13 of 17 For routine cultures, S. cerevisiae was grown on YEPD (2% glucose, 2% peptone, 1% yeast extract) or SC-Ura/ Leu medium (0.17% yeast nitrogen base, 0.5% ammo- nium sulfate, 2% glucose and uracil/leucine drop-out mix). The Escherichia coli strain used as plasmid host was DH5a. For selective growth, bacteria were grown on LB medium containing 100 mg/l ampicillin. Standard procedures were used for yeast genetic and DNA manipulations. DNA manipulation and plasmids General DNA manipulation methods were performed using standard techniques [83]. Whenever necessary, sequence verification of plasmid constructions was car- ried out on an automated 3730 DNA Analyzer (Applied Biosystems). The plasmid pJS05 [38] contains the promo- ter region of the gene MLP1 (YKL161c) fused to the NAT1 ORF (encoding for resistance to the antibiotic nourseothricin). Plasmids MLP1P-lacZ, CWP1P-lacZ and SED1P-lacZ including the promoter region of MLP1, CWP1 and SED1 genes fused to lacZ have been described previously [17]. Plasmids bearing the Slt2/Mpk1 alleles TA/YF (p2190), K54R (p2193) and the wild-type gene (p2188) were previously described [35]. Screening for yeast mutants displaying increased basal MLP1 expression Initially, the full collection of yeast mutant strains distrib- uted in seventy-five 96-well microtiter plates (Nunclon) were transformed with the plasmid pJS05 following a protocol of microtiter plate transformation [84], using SC-Ura as selection medium. A new set of plates contain- ing 100 μl of fresh SC-Ura medium were then inoculated with 5 μl of the original transformation cultures and allowed to grow at 28°C for 24 h to be used to inoculate the plates for the screening for MLP1 induction. Thus, 5 μl of these pre-inoculums were used to inoculate the definitive set of microtiter plates containing 95 μl of YEPD plus 300 μg/ml of nourseothricin (Werner BioA- gents, Germany) per well. The plates were incubated in a static culture at 28°C for 48-72 h and growth was deter- mined by measuring absorbance at 550 nm in each well with a microplate reader (Model 680, Bio-Rad). The para- meters described above, including incubation times, inoculum size and antibiotic concentration, were pre- viously optimized in order to prevent the growth of the wild-type strain (unaltered MLP1 expression). Nourseothricin susceptibility testing Selected yeast mutants and the wild-type strain, as control, were individually transformed with the plas- mid pJS05 following standard yeast genetic methods.