Pleiotropic roles of elongation factor P in Bacillus subtilis physiology revealed by phenotypic and multi-omics analyses.

UNLABELLED: Elongation factor P (EF-P) is conserved across the three kingdoms and alleviates ribosome stalling at the XPPX motif of a protein. A genome-wide ribosome-stalling atlas was previously created for the ancestral strain NCIB3610. However, reportedly, ribosome-stalling does not always decrease protein levels. To determine changes in protein levels in the efp mutant, we performed a proteomic analysis of the laboratory strain-based efp mutant. To evaluate its effects on translation, the amount of protein per mRNA molecule must be determined. Therefore, we performed the RNA-seq experiment. We identified 84 proteins containing the XPPX motif, whose translation required EF-P from 2,187 proteins detected in the proteomic analysis. The efp-dependent proteins included major sigma factor SigA, flagella ring component FliY, flagella motor protein MotB, thioredoxin TrxA, and manganese importer MntG. Western blotting revealed a decrease in the RNA polymerase (RNAP) subunits RpoB and RpoC in the efp mutant. The decrease in SigA and RNAP levels resulted in altered competition among core RNAP, SigA, and other Sigma factors, leading to a decrease in SigW. A decrease in FliY and MotB caused motility defects, and a decrease in TrxA resulted in a loss of heat tolerance. Moreover, a decrease in MntG altered the manganese requirement for growth at low temperatures. This study reveals the pleiotropic roles of EF-P in Bacillus subtilis cellular functions and sheds light on complex interactions between the transcriptome and proteome.

IMPORTANCE: Changes in the proteome have been reported to be consistent with those in the transcriptome. However, a serious change in a gene or protein, such as elongation factor P (EF-P), whose alteration causes pleiotropic effects, leads to proteomic changes that do not fully coincide with the transcriptome. The complex interactions between the transcriptome and proteome result in previously unknown and already known phenotypic changes in Bacillus subtilis. Changes in the expression of more than 1,000 genes were observed in the efp mutant, whereas changes in only approximately 200 proteins were observed when the same cut-off thresholds were used. This suggests that the robustness of the proteome against severe transcriptomic changes may require largely unknown post-transcriptional regulation.