Mids pNZ8901 pNZ8902 pNZ-xynA pNZ-usp45 pNZ-mntA pNZ-lmrA pNZ-nprE pNZ-bla pNZ-ycdH pNZ-xylP SURE expression vector, PspaSpn, CmR SURE expression vector, PspaSpn, EmR pNZ8902 carrying xynA of B. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone web subtilis [7] [7] [48] 168, amyE::spaRK, KanR, SURE expression system host [7] MG1363 derivative, pepN::nisRK [67] ReferencepNZ8902 carrying usp45 of L. lactis MG1363 [48] pNZ8902 carrying mntA of B. subtilis pNZ8902 carrying lmrA of L. lactis MG1363 pNZ8901 carrying nprE of B. subtilis pNZ8902 carrying bla of E. coli, pNZ8902 carrying ycdH of B. subtilis pNZ8902 carrying xylP of Lb. pentosus [48] [48] This work This work This work This workDNA microarray analysisThe overexpressed endogenous proteins were XynA, NprE, MntA and YcdH (Table 1). The overexpressed heterologous proteins were TEM-1 -lactamase from E. coli, Usp45 and LmrA (inactive mutant) from L. lactis and XylP from Lb. pentosus (Table 1). For the overproduction of the proteins, the SURE overexpression system was used [7]. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 The transcription profile of the control B. subtilis strain NZ8900 with empty pNZ8902 vector was compared to an isogenic target strain carrying one of the overexpression constructs: pNZ-xynA, pNZ-bla, pNZ-usp45, pNZ-mntA, pNZ-ycdH, pNZlmrA or pNZ-xylP. The target strain containing pNZnprE was compared to NZ8900 carrying empty pNZ8901. In total, eight independent microarray experiments were conducted. Strains harbouring overexpression constructs or the empty vectors pNZ8901 or pNZ8902 were grownTable 4 Oligonucleotides used in this studyOligo name nprE-fw nprE-rv bla_F bla_R ycdH-fw ycdH-rv xylP-fw xylP-rvaovernight in 10 ml TY broth supplemented with appropriate antibiotics and diluted the next day in 50 ml of fresh medium to an OD600 of 0.05. At an OD600 of 0.6, 0.1 (vol/vol) subtilin-containing supernatant of B. subtilis strain ATCC 6633 [73] was added to the growth medium to induce gene expression. After 30 min, 10 OD units of each culture were collected for RNA isolation. All the microarray experiments were performed in three biological replicates essentially as described before [74]. Total RNA was isolated using a High Pure RNA isolation Kit (Roche Applied Science). RNA quantity and quality were tested with a Nano Drop ND-1000 spectrophotometer (NanoDrop Technologies) and an Agilent Bioanalyzer 2100 (Agilent Technologies Netherlands BV), respectively. Amino allyl-modified cDNA was synthesized using the Superscript III Reverse Transcriptase Kit (Invitrogen), purified with the CyScribe GFX purification kit (Amersham Biosciences) and labeled with Cy3- or Cy5-monoreactive dye (Amersham Biosciences). Labeled cDNA was purified with the CyScribe GFX purification kit (Amersham Biosciences). Labeled cDNA concentration and dye incorporation were assessed with a Nano Drop ND-1000 spectrophotometer. The labeled cDNA was hybridized to oligonucleotide microarrays in Ambion Slidehyb #1 buffer (Ambion Europe Ltd) at 48 for 18?0 hours. Next, microarray slides were washed for 5 min in 2 ?SSC (300 mM NaCl, 30 mM sodium citrate) with 0.5 SDS, twice for 5 min in 1 ?SSC with 0.25 SDS and for 5 min in 1 ?SSC with 0.1 SDS, and dried by centrifugation. The slides were scanned with a GeneTac LS V confocal laser scanner (Genomic Solutions Ltd). ArrayPro 4.5 software (Media Cybernetics Inc., Silver Spring, Md., USA) was used to determine intensities of each spot on the microarrays using a local corners background correction method. Resulting expression levels were processed and normal.