predicted protein abundance of functionally related proteins co-evolve is reasonable, since the positive data was extracted from E.coli, which is a member of this subgroup. Therefore, it is likely that the functional interactions and general cellular mechanisms are more conserved within the g-proteobacteria. On the other hand, in archaea, the number of groups of organ- isms having functionally related pairs with signiﬁcantly higher correlation coefﬁcients than expected at random was much smaller. This may be due to several reasons. (i) The functional relations were based on E.coli as a reference, and these protein pairs are not all necessarily functionally related to the same extent in archaea. (ii) Codon bias has been suggested to be less associated with protein levels in archaea since most archaea are thermophiles, and other environmental factors can inﬂu- ence codon usage (36). (iii) Relatively fewer pairs of func- tionally related proteins occur in archaea, since not all the E.coli proteins are conserved in archaea. This results in a smaller sample size that can affect the P-values. In general, CAI and other measures of codon bias are only predictors of protein abundance levels. The correlation of abundant tRNAs with preferred codons in highly expressed genes has long been implied to improve the efﬁciency of translation (19,20). This led to the development of codon bias measures, which were shown to be correlated with the mRNA expression levels (17,25,37) and with protein abund- ance levels (14,15). However, it should be noted that these correlations were far from perfect, implying that only some of the variance in protein abundance can be explained by codon bias. Therefore, the results presented here are probably an underestimate of the phenomenon of correlated protein abund- ance levels in functionally related proteins. Examples of functionally related proteins with correlated profiles We describe here several examples of protein pairs that exhibit correlated proﬁles. The examples from the interaction dataset are pairs of proteins that have additional evidence of being translationally coordinated. The analysis was carried out for four-organism groups, as described in Figure 2 and in the Materials and Methods. However, for simplicity, the Spearman correlation coefﬁcients (rs) reported here were computed over groups of relevant organisms, and not over all the possible four-organism groups. (i) The a and b subunits of the F0F1–ATP synthase (COG0056 and COG0055). The F0F1–ATP synthase is one of the most highly conserved enzymes (38). It is composed of two components (F0 and F1) that use the proton gradient across the membrane in order to synthes- ize ATP. Three a subunits and three b subunits form a sphere that comprises the main portion of the F1 com- ponent (39). While these two proteins are similar at the protein sequence level (39), this does not necessarily imply that they are similar at the codon level. Indeed, a pairwise alignment of the coding sequences of the two proteins in E.coli found no signiﬁcant similarity. The two proteins are, for many genomes, within the same operon. In addition, they have been found to be translationally coordinated in the chloroplast of Chlamydomonas reinhardtii (40), and we would therefore expect that their protein abundance levels be generally correlated. Nucleic Acids Research, 2005, Vol. 33, No. 3 1055 Figure 3. CAI profiles within proteobacteria for the a and b subunits of the F0F1–ATP synthase (COG0056 and COG0055, respectively). Normalized CAI values for proteobacterial genomes are shown. For each organism, the normalization was based on the mean and standard deviation of all its proteins that appear in the COG database. The genomes represented here are Campylobacter jejuni (Cje), Neisseria meningitidis Z2491 (Nme), Agrobacterium tumefaciens (Atu), Brucella melitensis (Bme), Caulobacter vibrioides (Cvi), Mesorhizobium loti (Mlo), Rickettsia conorii (Rco), Rickettsia prowazekii (Rpr), Sinorhizobium meliloti (Sme), Escherichia coli K12 (Eco), Haemophilus influenzae (Hin), Pasteurella multocida (Pmu), Salmonella typhimurium (Sty), Vibrio cholerae (Vch), Xylella fastidiosa (Xfa) and Yersinia pestis (Ype). The connecting lines in the plot are for illustration only. Indeed, the overall correlation of these two CAI proﬁles (Figure 3) is high (rs = 0.85 over the proteobacteria and rs = 0.83 over the 34 organisms in which the subunits are present). Interestingly, the CAI values of both subunits are relatively low in the organisms Rickettsia conorii, Rickettsia prowazekii and Xylella fastidiosa. The former two are obligate parasites, while X.fastidiosa is a plant- pathogenic bacterium. Although codon usage has been suggested not to be a good estimator for protein abund- ance in these organisms (41,42), we did ﬁnd a signiﬁcant difference between the codon usage of ribosomal and other proteins in these organisms. A possible explanation for their low CAI values in this example lies is the fact that these organisms have been found to contain an ATP/ ADP translocase (COG3202), and thus they can import ATP from their host cells (43). This can explain a low cellular abundance of the F0F1–ATP synthase in these organisms. Interestingly, the other organisms within the ATP/ADP translocase COG do not have the a and b subunits. (ii) The b and b0 subunits of the DNA-directed RNA poly- merase (COG0085 and COG0086). The b and b0 subunits are highly conserved, and the genes encoding these sub- units are always adjacent (44). It has also been speculated that the assembly of these subunits in E.coli occurs co-translationally (45). Thus, it seems that the relative abundance of these proteins should be evolutionarily cor- related. Indeed, the correlation coefﬁcient between the