Abstract
BackgroundThere are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function. These proteins persist in databases with the annotation of a specific ‘putative’ function made by association with discernible features in the protein sequence.ResultsHere, we report the characterization of one such protein produced by the pathogenic soil bacterium Burkholderia pseudomallei, BPSL1375, which provided evidence for putative hemolysins in the COG3176 family to have experimentally validated hemolytic activity. BPSL1375 can be classified into the N-acyltransferase superfamily, specifically to members of the COG3176 family. Sequence alignments identified seven highly conserved residues (Arg54, Phe58, Asp75, Asp78, Arg99, Glu132 and Arg135), of which several have been implicated with N-acyltransferase activity in previously characterized examples. Using the 3D model of an N-acyltransferase example as a reference, an acyl homoserine lactone synthase, we generated 3D structure models for mutants of six of the seven N-acyltransferase conserved residues (R54, D75, D78, R99, E132 and R135). Both the R99 and R135 mutants resulted in a loss of hemolytic activity while mutations at the other five positions resulted in either reduction or increment in hemolytic activity.ConclusionsThe implication of residues previously characterized to be important for N-acyltransferase activity to hemolytic activity for the COG3176 family members of the N-acyltransferase provides validation of the correct placement of the hemolytic capability annotation within the N-acyltransferase superfamily.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0604-4) contains supplementary material, which is available to authorized users.
Highlights
There are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function
BPSL1375 is a member of the N-acyltransferase superfamily A systematic approach to characterize the functions of coding regions annotated as hypothetical proteins by Holden et al [27] resulted in the extraction of all such sequences followed by resubmission for sequence alignment based analysis. This included targeted searches for previously reported virulence and pathogenicity factors such as various hemolysins from other Gram negative bacteria. One of these searches identified motifs previously observed in the hemolysin of Ralstonia eutropha within the coding region of an Open reading frames (ORFs) originally annotated as encoding the hypothetical protein BPSL1375 by Holden et al [27]
A database search of the BPSL1375 sequence in GenBank revealed that the sequence contained domains that could be remotely associated with the N-acyltransferase (NAT) superfamily and shared sequence similarity to other proteins that were annotated as hypothetical proteins
Summary
There are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function. Burkholderia pseudomallei is a soil bacterium that causes melioidosis, a deadly disease endemic to South East Asia and Northern Australia [1]. This bacterium is recognized as a potential category B biothreat agent due to its aerosol infectivity and severe course of infection [2]. A toxin that deamidates and subsequently inactivates helicase activity of the translation initiation factor (eIF4a) has been reported [7]. Other aspects of this bacterium’s genome has been explored including the existence of small RNAs [8] and methylation of genomic DNA by restriction modification systems [9].
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