BMC Genomics 2008, 9:627 http://www.biomedcentral.com/1471-2164/9/627 to the loss of oxygen and those that more chronically respond to oxygen-dependent changes in cellular heme concentrations, for example those that are regulated by Hap1, Hap2/3/4/5, Rox1, Upc2 and Mot3 [6]. Overall, we expected and found that the same ESR/CER gene networks respond to poisoning of the respiratory chain under nor- moxia as to the shift to anoxia in the absence of inhibitor, networks that appear to be involved in balancing energy supply and demand, growth rate, and progression through the cell cycle. Results Statistical comparisons of the transcriptomic responses To compare the dynamics of the transcriptomic responses elicited by antimycin A treatment and anaerobiosis [6], we harvested samples after the same number of genera- tions following each treatment as previously we had shown such a growth-rate dependent alignment is supe- rior to time-dependent alignments ([5] see also [7]). Sep- arate ANOVAs were conducted to identify genes whose transcript levels responded significantly (P ≤ 0.01 after step-down Bonferroni adjustment) to each treatment. In total, 1754 genes were found to respond significantly at one or more time points to anaerobiosis, 901 to antimy- cin A treatment in air, and 669 to anaerobiosis in the pres- ence of antimycin A. Figure 1 compares the overall dynamics of each response expressed as the number of genes that were differentially expressed in each sample. Note that black bars indicate genes that responded significantly for the first time and gray those that had already responded at a previous time point yet continued to be differentially expressed with respect to the control. From this figure it is clear that the transcriptomic response to anaerobiosis in galactose medium is biphasic (Figure 1A) as was previously observed [6]. The first phase is comprised of a large set of genes (> 900) that exhibit an acute (0.04 – 0.08 genera- tions) yet transient (< 0.25 generations) response. After a considerable delay (≥ 0.25 generations), a second, smaller set of genes (> 400) is then differentially expressed, most for the duration of anaerobiosis. In comparison, the tran- scriptomic response to antimycin A treatment in air (Fig- ure 1B) is monophasic and, as anticipated, similar to the dynamics of the acute phase of the anaerobic response. Peak numbers of newly responding genes (black bars) appear at 0.08 generations, the same as for the acute anaerobic response (Figure 1A). Comparison of Figure 1C with 1A shows the presence of antimycin A drastically alters the anaerobic response; the large numbers of transiently responding genes observed in Figure 1A are conspicuously absent, leaving only those that respond after a substantial delay (2 generations). Interestingly, the dynamics of this response is remarkably similar to that elicited by anoxia in catabolite-repressed cells [5,6]. Taken together these results suggest that the acute, transient transcriptomic response observed in Fig- ures 1A and 1B is evoked by the inhibition of respiration, whether by chemical means (antimycin A) under nor- moxic conditions (Figure 1B) or the rapid removal of oxy- gen from respiratory competent cells (Figure 1A), whereas the delayed, chronic phase (Figures 1A and 1C) is invoked by oxygen deprivation. To further explore the composition of these networks, we divided the anaerobic gene set into those that responded acutely (≤ 0.25 generations) and those that responded more chronically (> 0.25 generations) and examined their overlap with those that responded to the antimycin A treatments. Figure 2 shows 67% of those that responded to the antimycin A treatment in air (light gray circle, left) exhibited an acute anaerobic response (bottom left) whereas 53% of those that responded to the subsequent shift to anaerobiosis (dark gray circle on right) exhibited a delayed anaerobic response (bottom right). Although the distinction between the acute and chronic phases is not absolute (Figure 2 open circles, lower portion), what is clear is that many of the same genes respond acutely to either anaerobiosis or antimycin A treatment in air. More- over, the acute response is abolished by adding antimycin A well before (3 generations) shifting the cells to anaero- biosis. To next determine if the same transcriptional net- works respond to these treatments, we separately clustered the temporal responses. Gene network discovery Gene networks were recovered using a novel bioinfor- matic approach [5,6] that uses SOM to cluster the tempo- ral profiles and then determines an appropriate level of granularity (number of clusters) by choosing that which results in the least likely configuration of transcription fac- tor binding sites (TFMs) among gene clusters as compared to random chance alone [see Additional files 1 and 2 and reference [6] for further details]. For the anaerobic data set, the clustered expression profiles are shown in Figure 3A (left heat map; anaerobic clusters C N2 1 – 13). The fig- ure also includes an abbreviated list of significantly (P ≤ 0.01) enriched TFMs (blue text to the left of each heat map) and overrepresented (P ≤ 0.01) MIPS functional cat- egories (right). Complete results, including statistics and gene-to-cluster membership, are provided in Additional files 3, 4, 5, 6. To facilitate direct comparisons of the two antimycin A treatments to anaerobiosis, Figure 3A not only shows the clustered expression profiles of genes that responded to anaerobiosis (N2, left heatmap) but the response of these same genes to antimycin A treatment in Page 3 of 14 (page number not for citation purposes)