Regulation of potassium-dependent Kdp-ATPase expression in the nitrogen-fixing cyanobacterium Anabaena torulosa

Abstract

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.

FIG. 1

Identification of Anabaena KdpB homolog. Proteins were extracted from A. torulosa cultures grown in N-supplemented (lanes 1 and 2) or N-deficient (lanes 3 and 4) conditions in BG-11/K5 (lanes 2 and 3) or BG-11/K0 (lanes 1 and 4) medium for 3 days. Equal amounts of protein (150 μg) were resolved by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (100 V for 1 h followed by 200 V for 3.5 h) and then electroblotted. The blots were allowed to cross-react with a primary anti-E. coli KdpB antiserum followed by a secondary anti-rabbit immunoglobulin G coupled to alkaline phosphatase.

FIG. 2

Cellular location of and effect of osmotic stresses on KdpB levels in Anabaena. (A) Nitrogen-fixing cultures were grown in BG-11/K5 (lanes 1 and 5) or BG-11/K0 (lanes 2 and 6) medium. On day 2, parts of the BG-11/K0 culture were stressed with either 0.2 M sucrose (lanes 3 and 7) or 0.1 M NaCl (lanes 4 and 8) for 24 h. Cellular extracts were separated into membrane (lanes 1 to 4) and cytosolic (lanes 5 to 8) fractions and the proteins were resolved by electrophoresis, as described for Fig. 1. C, medium controls; S, addition of sucrose; N, addition of NaCl. (B) Nitrogen-fixing cultures grown in BG-11/K5 (lane 2) medium for 2 days were subjected to 0.2 M sucrose (lane 3) or 0.1 M NaCl (lane 4) for 24 h. Membrane fractions were isolated and electrophoretically resolved for a longer duration (100 V for 1 h followed by 200 V for 4.5 h). Protein samples from E. coli MC4100 grown in BG-11/K0 medium for 6 h (lane 1) and from A. torulosa grown in BG-11/K0 for 24 h (lane 5) were included for comparison. Other details were as given for Fig. 1.

FIG. 3

Effect of osmotic and salinity stresses on KdpB levels in E. coli strain MC4100. Cells were grown either in K115 (lanes 1 and 5) or in BG-11/K0 (lanes 2 and 6) medium. Part of the cells grown in BG-11/K0 medium were stressed with either 0.4 M sucrose (lanes 3 and 7) or 0.25 M NaCl (lanes 4 and 8). Samples were collected at 6 (lanes 1 to 4) or 24 (lanes 5 to 8) h after the initiation of K⁺ deprivation. Other details were as given for Fig. 2B.

FIG. 4

The negative effect of exogenous K⁺ addition on KdpB levels in Anabaena. Cultures were grown in BG-11/K5 (lane 1) or BG-11/K0 (lane 2) medium for 2 days. On day 2, 5 mM KCl was added to part of the BG-11/K0 culture at time zero. Samples were removed 0.5 (lane 3), 2 (lane 4), 4 (lane 5), or 8 (lane 6) h after K⁺ addition, electrophoretically resolved for a longer duration, and processed for KdpB immunodetection as for Fig. 2B.