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Conservation analysis of the CydX protein yields insights into small protein identification and evolution.

Allen RJ, Brenner EP, VanOrsdel CE, Hobson JJ, Hearn DJ, Hemm MR - BMC Genomics (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Towson University, Towson 21252MD, USA. mhemm@towson.edu.

ABSTRACT

Background: 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. 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.

Results: Over 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.

Conclusions: This 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.

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Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [57].
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Fig5: Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [57].

Mentions: As an initial test of the functional importance of the CydX C-terminal region, a series of E. coli strains were constructed in which the amino acids following E25 were mutated in the cydX gene on the chromosome. The cydX genes in these strains were altered to encode either a six-serine tag at the C-terminus or a series of serines with flanking charged residues (Figure 5A). These residues were chosen in order to drastically alter the amino acid sequence while maintaining any general hydrophilic interactions within the periplasmic space and avoiding disruption of the orientation of the hydrophobic helix in the membrane. These strains were then tested for mutant phenotypes related to decreased CydX function, including mixed colony formation and sensitivity to β-mercaptoethanol. In all cases, the mutant CydX strains behaved like wild-type (unpublished data and Figure 5B), indicating that either there is no sequence specificity requirements for this region, the sequence requirements can be fulfilled by an essentially random series of hydrophilic amino acids, or this region of the protein is not required for CydX function under these conditions.Figure 5


Conservation analysis of the CydX protein yields insights into small protein identification and evolution.

Allen RJ, Brenner EP, VanOrsdel CE, Hobson JJ, Hearn DJ, Hemm MR - BMC Genomics (2014)

Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [57].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4325964&req=5

Fig5: Testing the functional importance of the CydX C-terminal amino acids. (A) Alignment of the E. coli CydX protein sequence along with six mutant sequences containing mutated C-terminal amino acid sequences. (B) Assay of CydX function was conducted using a zone assay testing the sensitivity to β-mercaptoethanol. Sensitivity was measured using zones of inhibition, and the diameter of the zone after addition of 10 μL of 12 M β-mercaptoethanol to a plate of bacteria is shown. The average and standard deviation of zone sizes was calculated from at least three replicate plates. Alignments were generated using the program MUSCLE [57].
Mentions: As an initial test of the functional importance of the CydX C-terminal region, a series of E. coli strains were constructed in which the amino acids following E25 were mutated in the cydX gene on the chromosome. The cydX genes in these strains were altered to encode either a six-serine tag at the C-terminus or a series of serines with flanking charged residues (Figure 5A). These residues were chosen in order to drastically alter the amino acid sequence while maintaining any general hydrophilic interactions within the periplasmic space and avoiding disruption of the orientation of the hydrophobic helix in the membrane. These strains were then tested for mutant phenotypes related to decreased CydX function, including mixed colony formation and sensitivity to β-mercaptoethanol. In all cases, the mutant CydX strains behaved like wild-type (unpublished data and Figure 5B), indicating that either there is no sequence specificity requirements for this region, the sequence requirements can be fulfilled by an essentially random series of hydrophilic amino acids, or this region of the protein is not required for CydX function under these conditions.Figure 5

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Towson University, Towson 21252MD, USA. mhemm@towson.edu.

ABSTRACT

Background: 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. 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.

Results: Over 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.

Conclusions: This 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.

Show MeSH
Related in: MedlinePlus