AIB transport by Rlv3841 and AA031 (Δ<i>npr</i>) and AA031 complemented with the empty vector pRK415 (EV), NPr, expressed from the constitutive lac promoter in multi-copy (+NPr) or a non-phosphorylatable variant (+NPrH17A).

<p>Data are averages (± SEM) from ≥3 independent cultures and rates are nmol min⁻¹ mg protein⁻¹.</p

AIB membrane transport by Rlv3841, PtsP107 (ptsP::Tn5) and various phosphocarrier protein mutants.

<p>Data are averages (± SEM) from 6 independent cultures and rates are nmol min⁻¹mg protein⁻¹.</p

AIB transport by Rlv3841 and PtsP107 (ptsP::Tn5) and PtsP107 complemented with the empty vector pRK415 (EV) or PtsP, expressed from the constitutive lac promoter in multi-copy (+PtsP).

<p>Additionally, two variants, +PtsPH367A and +PtsPΔGAF, were used for complementation. Data are averages (± SEM) from ≥3 independent cultures and rates are nmol min⁻¹ mg protein⁻¹.</p

Strains, plasmids and primers.

*<p>Restriction sites in primer sequences are underlined.</p

Effects of K⁺ limitation on ABC transport and growth of Rlv3841.

<p>(A) AIB transport by Rlv3841 at 1 µM and 1 mM KCl and PtsP107 as a control. Data are averages (± SEM) from 3 independent cultures and rates are nmol min⁻¹ mg protein⁻¹. (B) Generation times of Rlv3841 grown at 1 µM and 1 mM KCl with 10 mM NH₄Cl or 10 mM glutamate as the sole nitrogen source. Data are averages (± SEM) from 3 independent experiments with each 2 cultures. Data indicated with different letters are significantly different in a Student’s t-test (p<0.01).</p

In vitro phosphorylation of purified 6His-MBP-PtsP protein.

<p>(A) Example of a time course of 6His-MBP-PtsP phosphorylation. Each lane of the SDS-page contains ∼2 µg of protein and was incubated with 125 µM [³³P]PEP. The upper part shows the level of phosphorylation and the lower part the same gel after coomassie blue stain. (B) Phosphorylation of 6His-MPB-PtsP under various conditions. The phosphorylation is stable at 70°C for 10 min and boiled protein is inactive. The protein is not phosphorylated with 10 mM [³³P]ATP and also not in the presence of crude extract of RU4392 (Δask). (C) <i>In vitro</i> phosphorylation of purified 6His-MBP-PtsP and 6His-NPr protein. Lane 1–3 show phosphorylation of the single proteins and both proteins together with 125 µM [³³P]PEP. Lane 5–8 show a timecourse of the transphosphorylation. The upper part shows the level of phosphorylation and the lower part the same gel after coomassie blue stain.</p