Figure 5. Cellular levels of Rnk are insufficient to affect DksA function, but are similar to levels of GreB.

(A) The in vivo concentration of Rnk is lower than DksA and GreA, but comparable to GreB in log and stationary phase. Quantitative Western blots were performed on cell lysates of a wild-type strain (RLG5950) grown in MOPS medium (see Materials and Methods) in log phase (OD600~0.4) and stationary phase (OD600>4.5). Concentrations (fmol factor/µg total protein) were determined from at least 3 separate experiments (except for Rnk in stationary phase which was determined from 2 experiments).

(B) The rnk gene is dispensable for rRNA regulation in vivo. β-galactosidase activities (expressed in Miller Units) were determined in strains containing lacZ fused to rrnB P1. Promoter activities were assayed in wild-type (RLG3739) and Δrnk (RLG8255) cells growing in LB at 30°C in log phase (OD600~0.4 after ~4 generations of growth) and stationary phase (24 hr). β-galactosidase activities from ≥4 cultures were averaged and standard errors are shown.

(C). Rnk does not inhibit Gre factor function in tnaC expression in vivo. β-galactosidase activities (expressed in Miller Units) were determined in strains containing lacZ fused to the tnaC promoter region. Promoter activities were assayed in wild-type (RLG9166) and ΔgreAΔgreB (RLG9168) cells carrying an empty vector, pINIIIA1 (pRLG6332), or a vector expressing Rnk from the IPTG-inducible lpp-lac promoter, pRnk (pRLG9163). Cells were grown in LB at 30°C with 100 µg/ml ampicillin and 1 mM IPTG, and β-galactosidase activity was assayed in log phase (OD600~0.4 after ~4 generations of growth). β-galactosidase activities from ≥4 cultures were averaged and standard errors are shown. Similar trends were observed in stationary phase (date not shown)

(D). Rnk was overexpressed from the IPTG inducible lpp-lac promoter. Rnk levels were measured by Western blots performed on cell lysates from the cultures assayed for β-galactosidase activity described above. 40 ng of purified Rnk, ~39 µg of total protein from the cultures carrying the empty vector, pINIIIA1 (pRLG6332), and ~7.8 µg of total protein from the cultures carrying prnk (pRLG9163) were probed with anti-Rnk antibody. A representative blot is shown. Similar results were observed in stationary phase (data not shown).