BMC Genomics 2009, 10(Suppl 2):S4 http://www.biomedcentral.com/1471-2164/10/S2/S4 AMX CTC ENR 40 35 30 25 20 15 10 5 0 40 35 30 25 20 15 10 5 0 40 35 30 25 20 15 10 5 0 - C D E F G H I J K L M N O P Q R S T U V sSFiuoigmnumgrreaor3uypoefdsibgyniCficOanGtschanges in P. multocida gene expres- Summary of significant changes in P. multocida gene expression grouped by COGs. Numbers on the y-axis represent the percentage of genes in each COG category whose expression either significantly increased (black bars) or decreased (gray bars) in response to antibiotic treatment. COG category descriptions are: A, RNA processing and modification; K, Transcription; L, Replication, recombination and repair; D, Cell cycle control, mitosis and meiosis; V, Defense mechanisms; T, Signal transduction mechanisms; M, Cell wall/membrane biogenesis; N, Cell motility; W, Extracel- lular structures; U, Intracellular trafficking and secretion; O, Posttranslational modification, protein turnover, chaperones; C, Energy production and conversion; G, Carbohydrate transport and metabolism; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabo- lism R, General function prediction only; S, Function unknown; -, not in COGs cida, expression of genes encoding iron transporters fecC and fecE were reduced when it was grown in the presence of 1/4 MIC of AMX or ENR (Additional file 2, supplemen- tal table 2). Changes in the ratio of NAD+/NADH are known to influ- ence the levels of toxic superoxide formation, which ulti- mately leads to hydroxyl radical formation. NADH I levels are directly linked to the activities of TCA cycle enzymes that generate NADH from NAD+ [19]. ENR treatment caused decreased expression of sucB, and all three antibi- otics caused decreased lpdA expression, which could reduce hydroxyl radical formation. Expression of the gene encoding anaerobic respiration electron transfer enzyme molybdoenzyme dimethylsulfoxide reductase was reduced in response to AMX and ENR. AMX treatment reduced the expression of acetyl-CoA carboxylase enzyme (accB, accD), which catalyzes the first step of type II fatty acid biosynthesis as well as the step catalyzed by acyl car- rier protein S-malonyltransferase (fabD); by contrast, ENR increased the expression of these genes, which could result in increased fatty acid biosynthesis. DE genes: network analysis COG analysis of DE genes was useful for identifying over- all trends at a broad functional category level. Gene ontol- ogy (GO) analysis of P. multocida, which is available through Uniprot, was useful to some extent in adding spe- cific functions to the genes within each of the broad func- tional categories represented by COGs. However, neither of these two approaches could actually show exactly how the genes within a functional category were interacting with each other to bring about a specific function. To ena- ble network analysis, we used Pathway Studio to build interaction networks for all DE P. multocida genes with AMX, CTC and ENR. Careful analysis of these networks revealed expression cascades for some of the DE genes common to CTC and ENR. Expression of fnr increased in response to sub-MIC of CTC and ENR (Figure 4). Fnr regulates the transcription of many genes that are involved in either aerobic or anaero- bic respiration, including Nap nitrate reductase, which facilitates nitrate reduction in the periplasmic space. Expression of napD and napF increased in response to ENR and decreased with CTC (Additional file 3, supple- mental figure 1). NapD is a chaperone protein that is spe- cific for NapA and is required for the stability of NapA protein. NapF stimulates the Nap reductase. NapF pro- moters regulate the transcription of cmABCDEFGH operon in E. coli, which constitutes the cytochrome bio- genesis system. Expression of ccmH increased with CTC and decreased with ENR (Table 3). The identification of this cascade of gene regulation was possible by combining the network analysis with the functional information available at EcoCyc. The implications of up regulation of enzymes that facilitate anaerobic respiration in response to antibiotics remain unclear at present. Network analysis also facilitated the identification of dif- ferences in the SOS response with different antibiotics in P. multocida (Figure 5 (ENR) and Additional file 3, sup- plemental figures 2 (AMX) and 3 (CTC)). In particular, the response to ENR showed the signature gene expres- sion pattern typical of SOS response (Figure 5). Increased Page 5 of 10 (page number not for citation purposes)