Results EmhABC enhances growth at supra-optimal temperature Growth curves for P. fluorescens selleck chemical strains were determined at 10°C, 28°C or 35°C to allow sampling at the appropriate phase of growth in subsequent studies. The optimum growth temperature for wild type P. fluorescens LP6a is 28°C [15], 10°C is a growth-permissive sub-optimal temperature, and 35°C is ~2°C below the maximum growth temperature

of P. fluorescens LP6a wild type. Strains cLP6a and cLP6a-1 grown in seed cultures at 28°C were transferred to fresh medium and incubated at 10°C, 28°C or 35°C and growth was monitored Nepicastat research buy for 48 h. The growth curves of cLP6a and cLP6a-1, measured as OD600, were similar to each other at 10°C (Figure 1a) and at 28°C (Figure 1b). The lag phases of both cLP6a and cLP6a-1 were longer at 10°C than at 28°C

but the maximum OD600 achieved was greater at 10°C. The maximum OD600 achieved by cLP6a and cLP6a-1 was lower JPH203 purchase at 35°C and growth of the two strains was dissimilar (Figure 1c). The growth yield for strain cLP6a-1 at 35°C was about half that measured at 10°C and 28°C, and ~70% that of strain cLP6a at 35°C. Thus, disruption of emhABC in strain cLP6a-1 impaired its growth rate and cell yield at the supra-optimal temperature. Figure 1 Growth curves of P. fluorescens strains cLP6a and cLP6a-1. Growth of P. fluorescens strains cLP6a and cLP6a-1 at (a) 10°C, (b) 28°C or (c) 35°C determined as OD600 Each data point is the mean of three independent cultures, and error bars, where visible, Metalloexopeptidase indicate the standard deviation. Phenanthrene efflux by EmhABC is affected by incubation temperature To measure activity

of the EmhABC efflux pump, a rapid efflux assay [17] was performed using 14C-phenanthrene. In the efflux assay, suspensions of cLP6a and cLP6a-1 harvested at stationary phase were incubated with 14C-phenanthrene at a concentration below its aqueous solubility limit, to avoid any effects of dissolution on phenanthrene bioavailability. Partitioning of phenanthrene into the cells is very rapid, achieving steady state in less than 1 min [17]. At timed intervals, the radiolabel associated with the cell pellet is measured, and the steady state concentration is the sum of efflux and partitioning of phenanthrene. A significant increase in the concentration of phenanthrene associated with the cell pellet after addition of sodium azide indicates inhibition of active efflux, resulting in phenanthrene accumulation in the cell. A constant high concentration of phenanthrene in the pellet both before and after azide addition indicates absence of efflux.