A MarR family transcriptional regulator and subinhibitory antibiotics regulate type VI secretion gene clusters in Burkholderia pseudomallei.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2-lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6-lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2-lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.