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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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Related in: MedlinePlus

Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae. (C) Assay of complementation of the ΔcydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search (Burkholderia sp. 383), and an unrelated small protein (CydZ) from a different bacterial species. 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. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [57]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.
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Fig3: Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae. (C) Assay of complementation of the ΔcydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search (Burkholderia sp. 383), and an unrelated small protein (CydZ) from a different bacterial species. 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. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [57]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.

Mentions: To test the accuracy of our identification methods, we synthesized seven of the homologues identified in our screens and determined if they could functionally replace the CydX protein in E. coli by complementing the ΔcydX mutant. Four of these small proteins, identified in Actinobacillus pleuropneumoniae, Burkholderia xenovorans, Methylibium petroleiphilum PM1 and Pectobacterium atrosepticus were clear CydX homologues with significant Pfam hits (Figure 3A). One protein, encoded in Francisella philomiragia subsp. philomiragia ATCC25017, has a more divergent sequence but still returns a significant Pfam hit, while a sixth small protein, from Haemophilus influenzae 10810, has a divergent sequence and does not yield a Pfam hit (Figure 3A). The homologue from Cellvibrio japonicas Ueda107 was chosen as a representative of a few orphan homologues found to be encoded separately from a cydAB operon (Additional files 1 and 2a). We also tested a small protein identified by tblastn in Burkholderia sp. 383 that shows some sequence homology with CydX but lacks a significant Pfam hit and was ultimately scored as a negative, as well as an unrelated small Cyd protein identified in a cydAB operon in Klebsiella pneumoniae (Figure 3B). The ability of these small proteins to complement the E. coli ΔcydX mutant was assayed by transforming ΔcydX with a plasmid expressing each small protein, and testing the sensitivity of the transgenic strain to the reductant β-mercaptoethanol. Zone assays of these strains showed that all seven of the identified homologues complement the ΔcydX mutant, whereas the two negative control small proteins do not (Figure 3C). These results support the accuracy of our identification methods, provide evidence that the Pfam HMM for the CydX family is too stringent, and suggest that CydX homologues retain a similar functionality among divergent species.Figure 3


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)

Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae. (C) Assay of complementation of the ΔcydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search (Burkholderia sp. 383), and an unrelated small protein (CydZ) from a different bacterial species. 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. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [57]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.
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Related In: Results  -  Collection

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Fig3: Confirmation of functionality of CydX homologues. (A) Alignment of protein sequences of CydX homologues from Escherichia coli and other bacteria species. The small protein from Burkholderia sp. 383 (“Burkholderia383”) is not thought to be a homologue and was included as a negative control for the assay. Based on its significant sequence divergence was included in a separate alignment. (B) Alignment of the E. coli CydX protein with the CydZ protein from Klebsiella pneumoniae. (C) Assay of complementation of the ΔcydX β-mercaptoethanol sensitivity phenotype by expression of potential CydX homologues, a false positive from the tblastn search (Burkholderia sp. 383), and an unrelated small protein (CydZ) from a different bacterial species. 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. Species are as follows: Escherichia coli (“Escherichia”), Pectobacterium atrosepticus (“Pectobacterium”), Burkholderia xenovorans (“Burkholderia”), Actinobacillus pleuropneumoniae (“Actinobacillus”), Burkholderia sp. 383 (“Burkholderia sp. 383”), Klebsiella pneumoniae (“Klebsiella”), Cellvibrio japonicus Ueda107 (“Cellvibrio”), Methylibium petroleiphilum PM1 (“Methylibium”), Haemophilus influenzae 10810 (“Haemophilus”), and Francisella philomiragia subsp. Philomiragia ATCC 25017 (“Francisella”). Alignments were generated using the program MUSCLE [57]. Amino acids are colored based on their properties at physiological conditions as follows: red amino acids are hydrophobic, green residues are hydrophilic, purple residues are positively-charged and blue residues are negatively-charged. ‘*’ indicates that the residues are identical in all sequences and ‘:’ and ‘.’, respectively, indicated conserved and semi-conserved substitutions as defined by MUSCLE.
Mentions: To test the accuracy of our identification methods, we synthesized seven of the homologues identified in our screens and determined if they could functionally replace the CydX protein in E. coli by complementing the ΔcydX mutant. Four of these small proteins, identified in Actinobacillus pleuropneumoniae, Burkholderia xenovorans, Methylibium petroleiphilum PM1 and Pectobacterium atrosepticus were clear CydX homologues with significant Pfam hits (Figure 3A). One protein, encoded in Francisella philomiragia subsp. philomiragia ATCC25017, has a more divergent sequence but still returns a significant Pfam hit, while a sixth small protein, from Haemophilus influenzae 10810, has a divergent sequence and does not yield a Pfam hit (Figure 3A). The homologue from Cellvibrio japonicas Ueda107 was chosen as a representative of a few orphan homologues found to be encoded separately from a cydAB operon (Additional files 1 and 2a). We also tested a small protein identified by tblastn in Burkholderia sp. 383 that shows some sequence homology with CydX but lacks a significant Pfam hit and was ultimately scored as a negative, as well as an unrelated small Cyd protein identified in a cydAB operon in Klebsiella pneumoniae (Figure 3B). The ability of these small proteins to complement the E. coli ΔcydX mutant was assayed by transforming ΔcydX with a plasmid expressing each small protein, and testing the sensitivity of the transgenic strain to the reductant β-mercaptoethanol. Zone assays of these strains showed that all seven of the identified homologues complement the ΔcydX mutant, whereas the two negative control small proteins do not (Figure 3C). These results support the accuracy of our identification methods, provide evidence that the Pfam HMM for the CydX family is too stringent, and suggest that CydX homologues retain a similar functionality among divergent species.Figure 3

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