Figure 5.

MvaT oligomerization mutants are impaired for functionality in vivo. (A) Upper panel: Schematic of the ΔmvaT cupA1 lacZ reporter strain. Lower panel: Colony phenotypes of PAO1 ΔmvaT cupA1 lacZ cells (indicated ΔmvaT) carrying either the empty control vector pPSV37 (indicated -) or plasmids directing the IPTG-dependent synthesis of either MvaT-V (indicated MvaT), MvaT(F36S)-V [indicated MvaT(F36S)], or MvaT(R41P)-V [indicated MvaT(R41P)] grown overnight at 37°C on LB agar plates containing X-Gal and IPTG. The results show that phase-variable expression of the cupA genes that occurs in a ΔmvaT mutant strain is complemented by wild-type MvaT-V but not by MvaT-V mutants containing amino acid substitutions F36S or R41P. (B) ChIP experiment using wild-type and mutant forms of MvaT-V. PAO1 ΔmvaT cells carrying plasmids synthesizing MvaT-V (indicated WT) or MvaT(F36S)-V (indicated F36S) or MvaT(R41P)-V (indicated R41P) in an IPTG-inducible manner were grown to mid-log in LB in the presence of 1 mM IPTG, then cells were harvested for Western blot analysis and ChIP. Top panel: ChIP-enriched-DNA was measured by qPCR and fold enrichment determined by comparison to the PA2896 promoter as a non-binding control. Error bars represent 1 SD from the mean fold enrichment. (C) (Upper panel) Western blot analysis of MvaT-V (WT) (lanes 1–2) and mutant forms F36S (lanes 3–4) and R41P (lanes 5–6) probed with anti-VSV-G antibody. (Lower) Western blot analysis probed with antibody against the α subunit of RNAP serves as a control for sample loading.