Contributions of Zur-Controlled Ribosomal Proteins to Growth under Zinc Starvation Conditions

(A) Protein sequence alignment of the L33 C− proteins of B. subtilis (with the frameshift mutation corrected) and B. licheniformis (Bli). (B) Amino acid conservation of B. subtilis L33 C+ and C− paralogs. Black bars highlight the CXXC motif which binds a structurally important zinc cofactor in C+ proteins. (C) Northern blot of RNA isolated from CU1065 (wild type) and a zur mutant showing that the rpmGC transcript is only produced when Zur is absent. (D) Zur binds with high affinity to the rpmGC promoter in this EMSA using purified ZurBS protein, the rpmGC promoter, and a nonspecific (NS) promoter region with protein concentrations (in nM) increasing from left to right.

A zur mutation restores a wild-type growth rate to a strain lacking RpmGA and RpmGB only if the gene encoding RpmGC is corrected so as to encode a complete protein product (HB8251 [▵]). All other disruption combinations that do not restore the wild-type growth rate are shown as controls, with their strain number listed in parentheses (see Materials and Methods for genotypes). The rpmGC frameshift mutation is denoted “fs,” and the corrected rpmGC is denoted by “+”.

Mutations in zinc uptake and homeostasis proteins leads to growth defects in ZSMM. (A) Wild-type (✳) (WT) cells grow well in this medium. Cells defective for the ycdH-encoded ABC transporter grow more poorly (solid shapes), but there is no additional defect due to deletion of citM (6865 [•]), zinT (6867 [▴]), or both citM and zinT (6869 [▪]). In contrast, a ycdH yciC double mutant (HC) is significantly growth impaired (6866 [⋄]), and with the additional mutation of citM (6868 [○]), zinT (6890 [▵]), or both citM and zinT (6871 [□]) the growth defect phenotype now increases. (B) Only zinc is able to fully restore the growth defect of the HC double mutant observed in panel A. Growth at 12 h in ZSMM (see Materials and Methods) with 1 μM concentrations of added metals for the wild type (▪) and the HC double mutant strain (▦) was determined. The values shown are relative to the wild-type growth in each condition.

Zur-regulated r-protein paralogs contribute to growth under severe zinc starvation conditions (in the HC double-mutant background growing in ZSMM). Whereas the wild type (✳) grows well, ycdH yciC (6866 [⋄]) shows a reproducible lag and decrease in yield. This growth defect is magnified in the ycdH yciC ytiA (6882 [▴]), ycdH yciC yhzA (6880 [▪]), and ycdH yciC ytiA yhzA (6883 [⧫]) mutant strains.