aureus. Results YsxC is essential in S. aureus To test whether ysxC was essential in S. aureus, a strain containing a single chromosomal copy of ysxC under the control of a regulatable promoter (Pspac), repressed by LacI and requiring the inducer IPTG for expression was constructed
as indicated in Material and Methods (See also Figure 1). Growth of LC109 (SH1000 Pspac~ysxC/pGL485) at several IPTG concentrations (0 μM, 5 μM, 10 μM and 500 μM) was analysed on BHI agar plates supplemented with chloramphenicol to ensure maintenance of the lacI-containing plasmid (Figure 2A). Strong growth can be seen on the plate containing 500 μM IPTG with distinctive single colonies, which are absent on the plate without learn more IPTG. The phenotype on solid medium was further confirmed in liquid medium (Figure 2B). In a different
experiment it was shown that the presence or absence of IPTG does not affect growth of the wild type SH1000 strain (data not shown), while growth of LC109 (SH1000 Pspac~ysxC/pGL485) is IPTG concentration dependent (Figure 2B). No distinguishable alterations were observed on YsxC-depleted cells under light or transmission electron microscopy (data not shown). The number of viable counts on LC109 incubated in the absence of IPTG remained virtually unchanged, while in the presence of 1 mM IPTG it increased by 2 logs. Interestingly, even at 1 mM IPTG, LC109 (SH1000 Pspac~ysxC/pGL485) had a growth defect when compared to the wild type SH1000 strain (2.8×108 and 7.3×109 CFU after 7 h, respectively).
These results demonstrate that ysxC is apparently Paclitaxel order essential for growth of S. MLN0128 aureus in these conditions. Figure 1 Detailed scale representation of the P spac ~ ysxC (LC108/LC109) and ysxC ::TAP-tag (LC103) chromosomal constructs. λred recombination allowed highly specific chimera construction resulting in the Tet-T-Pspac or TAP-tag-kan cassette insertions. The relevant sequence junctions are shown for both constructs. Chromosomal sequence is shown in italics and relevant features generated by λred recombination are underlined. Figure 2 YsxC requirement for S. aureus growth. A) Strain LC109 (SH1000 Pspac~ysxC/pGL485) was grown on BHI agar plates containing 20 μg ml-1 Cam and 500 μM, 10 μM, 5 μM or 0 μM IPTG overnight. B) Exponentially growing cultures of strains SH1000 (●) and LC109 (SH1000 Pspac~ysxC/pGL485) (○,τ,ρ) were washed and resubcultured to approximately 1×106 CFU ml-1 in BHI (●) or in BHI supplemented with 20 μg ml-1 Cam plus different concentration of IPTG: 0 (○), 10 μM (▼) or 1 mM (△). Growth was monitored as CFU/ml. c) Western blot using anti-YsxC polyclonal antibodies. Strains SH1000 and LC109 (SH1000 Pspac~ysxC/pGL485) were grown to an OD600 = 0.5 in BHI and BHI plus 20 μg ml-1 Cam, respectively. Cells were harvested by centrifugation, the membrane protein fraction extracted and MM-102 datasheet samples were separated by 12% (w/v) SDS-PAGE.