NapM, a new nucleoid-associated protein, broadly regulates gene expression and affects mycobacterialresistance to anti-tuberculosis drugs

To identify the genes with napM-dependent differential expression, the gene expression profiles of the wild-type M. smegmatis strain and the napM-deleted strains were compared. For this case, the genes with fold changes exceeding 2 (P < 0.05) were considered significant. Strikingly, 156 genes were found to be differentially expressed in the absence of napM. Among these genes,121 were upregulated and 35 were downregulated. For microarray studies, all M. smegmatis and M. smegmatis napM::hyg strains were prepared in triplicate (OD600 = 1.2). RNAs were isolated as described previously (Yang et al., 2012), purified using an RNeasy mini kit (Cat#74106, QIAGEN, GmBH) according to the manufacturer’s instructions, and checked for an RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Total RNAs were processed in microarray facilities affiliated with Sangon Biotech (Shanghai) Co., Ltd. and hybridized to arrays that consisted of 665060-mer probes in situ synthesized by Agilent Technologies. The probes were designed in accordance with the genome sequences of Mycobacterium smegmatis mc2 155(GenBank accession numbers: NC_008596.1), which include 6938 open reading frames. The scanned output files were analyzed with the Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) and Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). The genes that showed significant differences in expression levels (P ≤ 0.05) and fold changes exceeding 2.0 between M. smegmatis and the M. smegmatis napM::hyg strain were identified and noted as marked gene-expression changes for further analysis.