The sample Cy5-dye labelled cDNAs and the reference Cy3-dye labeled cDNAs were mixed (1:1) and purified for removal of uncoupled dye by using a QIAquick PCR purification kit (Qiagen, Valencia, CA), as described by the supplier. The pellets obtained were dissolved in 35 μl hybridization buffer (5x SSC, 0.2% SDS, 5x Denhardt’s solution, 50% (v/v) formamide and 0.2 ug/ul denatured herring-sperm DNA), boiled for 5 min and spun down briefly. Networks construction and analysis A bipartite

network, named Network 1 was constructed Selleckchem AZD6738 with the novo generated gene expression data in this study by connecting two sets of nodes: one set was formed by genes differentially transcribed under several culture conditions. The other set of nodes included the environmental conditions (heat, find more oxidative and acid stress in anoxic and oxic condition, osmotic stress under anoxic condition and non-stressing anoxic conditions) Selleck Anlotinib combined with the regulation pattern, i.e. up or down-regulation. Network 2 was constructed by extending network with nodes representing genes and conditions to include the transcriptional response reported during the lag period,

exponential growth and stationary phase [7] and in immobilized cultures in different stages [8, 9]. Network 3 was a bipartite genome scale network including all genes in the genome of S. Typhimurium LT2 and plasmids of S. Typhimurium SL1344 as previously described [10]. Edges connected two sets of nodes. Genes constituted one of these sets of nodes. The genome composition was obtained from the Genome Project NCBI database [65]. The other set of nodes included metabolic pathways and cellular functions, according to the KEGG database [66], the CMR-TIGR database [67] and the COGs (Clusters of Orthologous Groups of proteins) functional categories obtained from the Genome Project NCBI database [65]. The number of nodes was 5153, from

which 4717 were genes and the remaining 436 nodes represented metabolic pathways and cellular functions. There were 11626 edges between these two sets of nodes. For networks representation and topological quantification we used the programs PAJEK [68] and Cytoscape [69]. Networks modularity was estimated implementing CYTH4 the fast modularity maximization algorithm [11]. Cluster analysis Hierarchical clustering was performed using the SAS 9.2 software [70] on the novo generated microarray data in this work using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Expression values were coded as 1 if genes were induced, -1 if repressed and 0 if not affected. Environmental conditions (heat, oxidative and acid stress in anoxic and oxic condition, osmotic stress under anoxic condition and non-stressing anoxic conditions) were clustered according to the gene expression values. Construction of mutants Cultures were grown in LB broth (Oxoid, CM1018) or on solid media consisting of LB-broth with addition of 1.