The Multiple Roles of Hypothetical Gene BPSS1356 in <i>Burkholderia pseudomallei</i>

<div><p><i>Burkholderia pseudomallei</i> 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 <i>B. pseudomallei</i> 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 <i>B. pseudomallei</i> strains and present only in the <i>Burkholderia</i> 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.</p></div

PCR screening result of mutant candidates.

<p>The PCR amplicon of 2.2(Lane 2). This indicates that mutant (Lane 1) was obtained.</p

Identities of RpoC-N interactive proteins with locus tags and their COG annotations.

<p>Identities of RpoC-N interactive proteins with locus tags and their COG annotations.</p

Growth kinetics of wild type <i>B. pseudomallei</i> and ΔBPSS1356 mutant strains when grown using glycerol as the sole carbon source.

<p>The ΔBPSS1356 mutant strains showed greater aggregation after 60 hours of incubation.</p

Growth rate of wild type <i>B. pseudomallei</i> K96243 and ΔBPSS1356 mutant strains in LB broth medium.

<p>Mutant strain exhibited less cell density during stationary growth phase.</p

Biofilm formation analysis of wild type <i>B. pseudomallei</i> and ΔBPSS1356 mutant strains in LB.

<p>The ΔBPSS1356 mutant exhibited decreased biofilm formation.</p

Growth kinetics of wild type <i>B. pseudomallei</i> and ΔBPSS1356 mutant strains when grown using high salt medium.

<p>The ΔBPSS1356 mutant showed a reduced growth compared to the wild type after 8 hours of incubation.</p

Oxidative stress assay of wild type <i>B. pseudomallei</i> and ΔBPSS1356 mutant strains.

<p>Both strains showed no significant difference in toleration to oxidative stress.</p

Scanning electron microscopy of (A) wild type <i>B. pseudomallei</i> and (B) ΔBPSS1356 mutant strains.

<p>Mutant strain showed rougher cell surface.</p

Transmission electron microscopy of (A) wild type <i>B. pseudomallei</i> and (B) ΔBPSS1356 mutant strains.

<p>Mutant showed shrunken cytoplasm.</p