Bohlin et al. BMC Genomics 2010, 11:464 http://www.biomedcentral.com/1471-2164/11/464 Page 4 of 8 p-value 0.05 0.003 <0.001 0.002 <0.001 <0.001 <0.001 0.001 0.04 <0.001 <0.001 <0.001 0.002 0.01 <0.001 <0.001 Table 1 The coefficient estimates from the GCVAR regression model Factor Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Phylum Oxygen GC Category Acidobacteria Actinobacteria Bacteroides Betaproteobacteria Chlamydiae Crenarchaeota Cyanobacteria Deltaproteobacteria Epsilonproteobacteria Euryarcheota Firmicutes Gammaproteobacteria Planctomycetes Spirochaetes Anaerobic - Chromosomes 2 42 16 64 8 16 17 18 12 31 89 92 1 11 - - Average %GC 60 66 44 64 43 48 48 58 38 46 37 47 55 37 - - Average size (mbp) 7.8 4.8 3.6 3.4 1.9 2 4.4 4.7 1.9 2.4 2.6 3.7 7.2 1.7 - - Coefficient estimate -0.23 -0.11 0.18 0.1 -0.28 0.22 0.3 0.15 0.1 0.16 0.12 0.12 -0.48 0.14 0.11 0.37 The variable GC is continuous while phylum and oxygen are categorical variables. Note that for the phylum variable we have used the sum-to-zero parameterization, i.e. all estimated effects are deviations from the mean phylum effect. For the oxygen requirement variable however, we used a relative parameterization where the category “aerobic” is the reference, i.e. the estimated effect is the deviation from the aerobic effect. In addition, the number of chromosomes, average %GC, and average genomes size in mbp, are included for each phylogenetic group. with mean genomic GC content, oxygen requirement, habitat, optimal growth temperature, and genome size as predictors. In addition, an interaction term between GC content and phylum was added to account for more similar GC content within phyla (Equation (5) in the Methods section). Using this regression model we found that oxygen requirement was the only significant factor (p < 0.001). GCVAR was significantly higher in the gen- omes of anaerobic microbes (103 chromosomes) as compared to the genomes of aerobic microbes, meaning that the genomes of anaerobic microbes tend to have a more heterogeneous distribution of GC content than genomes of aerobic microbes (246 chromosomes). Facultative microbes were found to have GCVAR values in the region between aerobic and anaerobic microbes (see Figure 2). The associations between mean genomic GC content, GCVAR and oxygen requirement The regression models described above indicates that aerobic microbes have genomes with more homoge- neous GC content than those of organisms with faculta- tive and anaerobic oxygen requirement (see Figure 2). It has been shown that GC rich genomes tend to be more homogeneous in terms of base composition than AT rich genomes [18,19,34]. Aerobic microbes have been associated with GC rich genomes [8]. This result is sup- ported by our linear regression model only when we ignore phylogenetic bias is (p < 0.001). However, adding phyla as a predictor (Equation (5) in the Methods sec- tion) fails to demonstrate such an association (p ~0.9). We found that mean genomic GC content was asso- ciated with GCVAR, but there was no linear relationship between mean genomic GC content and GCVAR (Figure 3), although this does not exclude a non-linear relationship. GCVAR and DNA uptake There are many indications that mean genomic GC con- tent is as much affected by the environment as by phyla [10-13]. It is also well known that chromosomally inte- grated foreign DNA may differ in base composition as compared to host DNA. The difference in base compo- sition between foreign and host DNA is assumed to be the result of exposure to different selective pressures. It is thought that such genetic regions may be acquired from horizontal transfer or other means of DNA uptake [6,24-26]. Since pathogenesis is often associated with horizontally transferred DNA, i.e. pathogenicity islands, [27], establishing a link between any genomic property and horizontal transfer is of considerably interest. How- ever, no significant association (p ~ 0.25) was found between the dichotomous pathogenicity factor and GCVAR using the regression model that included all covariates discussed above (Equation (3) in the Methods section). Base composition and oxygen requirement The introduction of atmospheric oxygen is presumed to have had profound effects on environment and life [35]. Increase in atmospheric oxygen is believed to have influ- enced cellular compartmentalization and thus to have