Validation of a Burkholderia pseudomallei Hypothetical Protein and Determination of Its Translational Start Codon Using Chromosomal Integration of His-Tag Coding Sequence

Yam, Hokchai; Rahim, Ainihayati Abdul; Luan, Ooi Gim; Samian, Razip; Manaf, Uyub Abdul; Mohamad, Suriani; Najimudin, Nazalan

doi:10.1007/s10930-012-9398-5

Cited by 2 publications

(2 citation statements)

References 12 publications

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“…The start codon of BPSS1356 was verified by integrating an in-frame C-terminal His-Tag coding sequence into the chromosomal copy of BPSS1356 [26] . The C-terminal His-tagged BPSS1356 protein produced by resultant strain was then purified and subjected to an N-terminal sequencing analysis which subsequently revealed its start codon.…”

Section: Discussionmentioning

confidence: 99%

The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei

et al. 2014

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Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β′ subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.

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Section: Discussionmentioning

confidence: 99%

The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei

et al. 2014

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“…Open reading frames (ORFs) annotated as hypothetical proteins make up almost 25 % of the B. pseudomallei genome [ 26 ]. Cruz-Migoni et al [ 7 ] reported a genome-wide project to characterize B. pseudomallei hypothetical proteins in an attempt to discover proteins with novel functions, which may include putative virulence factors.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Burkholderia pseudomallei protein BPSL1375 validates the Putative hemolytic activity of the COG3176 N-Acyltransferase family

et al. 2015

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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.

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Validation of a Burkholderia pseudomallei Hypothetical Protein and Determination of Its Translational Start Codon Using Chromosomal Integration of His-Tag Coding Sequence

Cited by 2 publications

References 12 publications

The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei

The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei

Characterization of Burkholderia pseudomallei protein BPSL1375 validates the Putative hemolytic activity of the COG3176 N-Acyltransferase family

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