Next we compared the growth rates of the ΔksgA strain and the par

Next we compared the growth rates of the ΔksgA strain and the parental RN cells. We grew both strains in liquid media at a variety of temperatures (Additional file 1) and calculated the doubling times for each strain, shown in Table  2. The strains grew at similar rates at 30°C, 37°C, and 42°C. However, at the lower temperatures

of 25°C and 15°C the ΔksgA strain grew significantly slower than the RN strain. We can conclude from these data that while knockout of ksgA does not affect cell growth using our test conditions at and around human physiological temperatures the cells become cold-sensitive upon loss of KsgA. Table 2 Doubling times of RN4220 and ΔksgA strains   MCC950 purchase Doubling time (min)   RN4220 ΔksgA 15°C 408.2 ± 18.2 473.0 ± 17.2 25°C 82.1 ± 4.1 93.4 ± 2.0 30°C 48.5 ± 0.6 50.2 ± 2.2 37°C 39.2 ± 1.8 39.4 ± 1.7 45°C 50.6 ± 1.5 54.3 ± 3.5 We then performed polysome analysis of the ribosomal selleck chemical particles of both strains to ascertain the effects of ksgA knockout on ribosome biogenesis. In these experiments ribosomal material is separated into mature, functional 70S ribosomes and free 30S and 50S subunits. In this way we can visualize increases in immature subunits as a portion of the total ribosomal material. As shown in Figure  2, knockout of ksgA did not result in a significant increase in relative amounts of free

30S subunits. Polysome profiles of MLN2238 nmr the RN and ΔksgA strains were similar at 42°C, 37°C, and 25°C; the proportion of free subunits increased with lowering temperature in both strains. Figure 2 Polysome analysis of the RN4220 and ΔksgA strains. Each chromatogram was normalized to a value of 1.0 for the 70S peak; successive chromatograms were offset by 0.2 on the y-axis. Our laboratory previously observed that knockout of ksgA in E. coli led to a difference Etofibrate in sensitivity to aminoglycoside antibiotics [9]. Specifically, the ΔksgA strain was more sensitive to the 4,6 class of aminoglycosides and less

sensitive to 4,5-aminoglycosides, with no change in sensitivity to the aminoglycoside streptomycin. We performed a similar experiment in S. aureus, growing the RN and ΔksgA strains on increasing amounts of the antibiotics kanamycin (a 4,6 aminoglycoside), paromomycin (a 4,5-aminoglycoside) and streptomycin (Table  1). The ΔksgA strain was more sensitive to both kanamycin and paromomycin, with no change in sensitivity to streptomycin. Overexpression of catalytically inactive KsgA is deleterious Overexpression of KsgA, as well as a catalytically inactive mutant of KsgA, was deleterious to E. coli growth rates under a variety of conditions [3]. In order to see if these results extended to S. aureus we cloned the ksgA gene from the RN4220 strain and constructed the equivalent mutation, E79A. We expressed both WT and E79A protein in RN and ΔksgA cells, using the empty vector (pCN) as a control.

Comments are closed.