Gene transcript BMEII0051 was found to be down-regulated 1.9 Nutlin-3 and 2.8-fold in response to a vjbR deletion and addition of C12-HSL to wildtype cells (respectively) at an exponential growth phase (Table 2). This luxR-like gene is located downstream of a VjbR consensus promoter sequence and thus most likely directly promoted by VjbR . The second luxR-like gene, BMEI1607, was up-regulated 1.8-fold and 3.0-fold in the vjbR mutant and in response to exogenous C12-HSL at the exponential growth phase (respectively), and down-regulated 1.5-fold by the deletion of vjbR at the stationary
growth phase (Table 2). This gene locus was not found to be located downstream of a predicted VjbR promoter sequence and may or may not be directly regulated by VjbR. Additionally, blxR was found to be induced 27.5-fold in wildtype cells treated with C12-HSL at the stationary growth phase by qRT-PCR (Table 1). Likewise, qRT-PCR verified BGJ398 in vitro a 2.9-fold down-regulation of vjbR in wildtype cells supplied with exogenous
C12-HSL at the stationary growth phase. The identification and alteration of genes containing the HTH LuxR DNA binding domain by ΔvjbR and C12-HSL administration, particularly one located downstream of the VjbR consensus promoter sequence, is of great interest. These observations potentially suggest a hierarchical arrangement of multiple transcriptional circuits which may or may not function in a QS manner, as observed in organisms such as P. aeruginosa . AHL synthesis. The deletion of vjbR or addition of C12-HSL resulted in alteration in the expression of 15 candidate AHL synthesis genes, based on the gene product’s potential to interact with the known metabolic precursors of AHLs, S-adenosyl-L-methionine (SAM) and acylated acyl carrier protein (acyl-ACP) (Additional File 2, Table S2) . An E. coli expression system was utilized because B. Methocarbamol melitensis has been shown to produce an AiiD-like lactonase capable of inactivating C12-HSL . Cross streaks with E. coli AHL sensor strains and clones expressing
candidate AHL synthesis genes failed to induce the sensor stains, while positive control E. coli clones expressing rhlI and lasI from P. aeruginosa and exogenous 3-oxo-C12-HSL did in fact induce the sensor strains (data not shown) . C12-HSL regulates gene expression independent of VjbR In addition to the investigation on the influences of a vjbR deletion or addition of C12-HSL to wildtype bacteria on gene expression, treatment of ΔvjbR with exogenous C12-HSL was also assessed by microarray analyses. Compared to untreated wildtype cells, 87% fewer genes were identified as differentially altered in response to C12-HSL in the vjbR null background as opposed to wildtype cells administered C12-HSL.