Abstract
BackgroundThe reliable identification of proteins containing 50 or fewer amino acids is difficult due to the limited information content in short sequences. The 37 amino acid CydX protein in Escherichia coli is a member of the cytochrome bd oxidase complex, an enzyme found throughout Eubacteria. To investigate the extent of CydX conservation and prevalence and evaluate different methods of small protein homologue identification, we surveyed 1095 Eubacteria species for the presence of the small protein.ResultsOver 300 homologues were identified, including 80 unannotated genes. The ability of both closely-related and divergent homologues to complement the E. coli ΔcydX mutant supports our identification techniques, and suggests that CydX homologues retain similar function among divergent species. However, sequence analysis of these proteins shows a great degree of variability, with only a few highly-conserved residues. An analysis of the co-variation between CydX homologues and their corresponding cydA and cydB genes shows a close synteny of the small protein with the CydA long Q-loop. Phylogenetic analysis suggests that the cydABX operon has undergone horizontal gene transfer, although the cydX gene likely evolved in a progenitor of the Alpha, Beta, and Gammaproteobacteria. Further investigation of cydAB operons identified two additional conserved hypothetical small proteins: CydY encoded in CydAQlong operons that lack cydX, and CydZ encoded in more than 150 CydAQshort operons.ConclusionsThis study provides a systematic analysis of bioinformatics techniques required for the unique challenges present in small protein identification and phylogenetic analyses. These results elucidate the prevalence of CydX throughout the Proteobacteria, provide insight into the selection pressure and sequence requirements for CydX function, and suggest a potential functional interaction between the small protein and the CydA Q-loop, an enigmatic domain of the cytochrome bd oxidase complex. Finally, these results identify other conserved small proteins encoded in cytochrome bd oxidase operons, suggesting that small protein subunits may be a more common component of these enzymes than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-946) contains supplementary material, which is available to authorized users.
Highlights
The reliable identification of proteins containing 50 or fewer amino acids is difficult due to the limited information content in short sequences
Homologues using tblastn and a hidden Markov model-based screen In order to investigate the extent of CydX conservation, complete genomes from 1095 taxa that span the major Eubacterial divisions were screened for potential homologues of the E. coli CydX protein
The first technique was a series of searches for CydX homologues in each genome using the protein-nucleotide sixframe translation Basic Local Alignment Search Tool for microbial genomes, with the E. coli CydX protein sequence used as the query for each search
Summary
The reliable identification of proteins containing 50 or fewer amino acids is difficult due to the limited information content in short sequences. The 37 amino acid CydX protein in Escherichia coli is a member of the cytochrome bd oxidase complex, an enzyme found throughout Eubacteria. Little attention has been paid to open reading frames coding for small proteins of fewer than 50 amino acids (aa), but in the last decade, small proteins have been discovered and associates with the CydAB subunits of the complex and is required for oxidase activity [6,7]. Cytochrome oxidases are a broad family of integral membrane complexes that catalyze the terminal electron transfer in eubacterial and archaeal respiration [8]. These complexes couple the oxidation of either quinol or cytochrome c substrates with the reduction of O2 to water (Figure 1B). Cytochrome bd is under investigation as an antibiotic target [9]
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