BMC Genomics 2009, 10(Suppl 2):S4 http://www.biomedcentral.com/1471-2164/10/S2/S4 Table 3: Fnr regulated genes and their response to sub-MIC of chlortetracycline and enrofloxacin * Ratio of treatment/control normalized intensities. ns: no significant change in gene expression with sub-MIC of antibiotic. GeneID 1244015 1243352 1243353 1243356 1243357 1243359 1243360 1244939 1244940 1244941 1244945 Product Name Fumarate/nitrate reduction transcriptional regulator Cytochrome c biogenesis protein CcmA CcmB cytochrome c-type biogenesis protein CcmE CcmF CcmH CcmH Hypothetical protein PM1592 NapD Nitrate reductase NapC Conclusion Our global transcriptome analysis of P. multocida response to three antibiotics with differing modes of action identi- fied gene expression changes in a little over a quarter of the annotated open reading frames in the genome. The antibiotics had varying effects on various cellular and met- abolic functions. Amoxicillin reduced the overall tran- scription rate as reflected by the majority of the identified significant changes in gene expression showing a down- ward trend in expression. An interesting aspect of the AMX response was the marked lack of expression of SOS response genes. Enrofloxacin, a DNA gyrase inhibitor, SeFOnigrSoufrloeesxp5aocinnse gene network in P. multocida in response to SOS response gene network in P. multocida in response to enrofloxacin. P. multocida sub-MIC ENR response was marked by significant changes in SOS response genes. Red nodes are genes with increased expression and green nodes are genes with decreased expression. Pink nodes represent no significant changes in gene expression while gray nodes are orthologs from gram-negative species in the molecular interaction database in Pathway Studio. Locus fnr ccmA ccmB ccmE ccmF ccmH_1 ccmH_2 napF napD napA napC CTC* 1.49 -0.70 -0.45 -0.77 ns 1.41 1.56 -0.50 -0.52 -0.81 1.40 ENR* 1.43 1.80 1.27 -0.57 -0.44 -0.43 -0.52 2.11 1.35 -0.76 -0.68 resulted in the significant overexpression of its target gene and induced a typical SOS response. Analysis of DE genes in the context of interacting protein networks facilitated the identification of coordinated regulation of gene expression across different COG functional categories. Sub-MIC of antibiotics influenced the expression of viru- lence factors in P. multocida. Our results form the frame- work for understanding the global effects of antibiotics on P. multocida, which will aid rational drug design for con- taining as well as treating infections caused by this patho- gen across multiple species. Methods Bacterial culture and RNA isolation P. multocida Pm70 was cultivated in brain heart infusion broth (BHI) at 37°C with rotary aeration. Minimum inhibitory concentrations (MICs) of AMX, CTC and ENR for Pm70 are 0.5 g/ml, 4 g/ml, and 0.031 g/ml, respec- tively [10]. Growth kinetics of Pm70 in the presence of 1/ 4 MIC of the three antibiotics were previously described [10]. Stationary phase cultures of Pm70 were used to inoc- ulate 50 ml BHI to an initial A600 of 0.05; antibiotic treated cultures contained 1/4 MIC of AMX, CTC, or ENR, and control cultures were grown without antibiotics. For each treatment, cultures were grown in triplicate to mid- log phase (A600 of 0.8). RNAprotect (Qiagen) was added to culture samples, and bacteria were harvested by centrif- ugation (10,000 × g, 10 min, 4°C). Pellets were stored at -80°C. Total RNA from three biological replicate cultures (for untreated control culture as well as antibiotic treated cul- tures) was isolated using Qiagen RNeasy kit using the manufacturer's protocol. The quality and concentration of RNA was determined by Agilent Bioanalyzer. Microarrays and data analysis A custom oligonucleotide microarray consisting of all the 2015 annotated open reading frames (ORFs) of Pasteurella Page 7 of 10 (page number not for citation purposes)