Synonymous Mutations in rpsT Lead to Ribosomal Assembly Defects That Can Be Compensated by Mutations in fis and rpoA

Abstract

We previously described how four deleterious synonymous mutations in the Salmonella enterica rpsT gene (encoding ribosomal protein S20) result in low S20 levels that can be compensated by mutations that restore [S20]. Here, we have further studied the cause for the deleterious effects of S20 deficiency and found that the S20 mutants were also deficient in four other 30S proteins (S1, S2, S12, and S21), which is likely due to an assembly defect of the S20 deficient 30S subunits. We examined the compensatory effect by six additional mutations affecting the global regulator Fis and the C-terminal domain of the α subunit of RNA polymerase (encoded by rpoA). The fis and rpoA mutations restored the S20 levels, concomitantly restoring the assembly defect and the levels of S1, S2, S12, and S21. These results illustrate the complexity of compensatory evolution and how the negative effects of deleterious mutations can be suppressed by a multitude of mechanisms. Additionally, we found that the mutations in fis and rpoA caused reduced expression of other ribosomal components. Notably, some of the fis mutations and the rpoA mutation corrected the fitness of the rpsT mutants to wild-type levels, although expression of other ribosomal components was reduced compared to wild-type. This finding raises new questions regarding the relation between translation capacity and growth rate.

The coordination of replication initiation with growth rate in Escherichia coli

“…The proteomic analyses were performed as described in Knöppel et al, (2020). Briefly, cultures were started by inoculating a 50 ml succinate medium with 250 μ l overnight culture.…”

“…The pellets were resuspended in 50 ml pre-warmed succinate medium and allowed to continue growing for 90 or 180 minutes before being pelleted, washed twice in PBS, and frozen. The proteomics core facility at the Sahlgrenska Academy, Gothenburg University, homogenized the samples, digested with trypsin and labeled the peptides with TMT 10-plex isobaric tagging reagents, and performed relative quantification of peptides by LC-MS/MS, as described in Knöppel et al, (2020).…”

Evaluation of conventional methods for species identification of Staphylococcus aureus using MALDI-TOF MS, protein identification and quantification

“…Mutations of rpsT were defective in many steps of translation initiation and ribosomal assembly. 31 In this study, the infections from S. aureus. imply more virulence than those from S. argenteus, S. hominis and S. haemolyticus.…”

Proteomic Profiling of Outer Membrane Vesicles Released by Escherichia coli LPS Mutants Defective in Heptose Biosynthesis

“…However, the downregulation of stress-related proteins such as PspA and YdiY [ 41 , 42 ] is likely attributable to their relationship with OMV protein construction, and the suppression of the aforementioned gene transcripts or protein expression by OMV protein knockout. Moreover, upregulation of the ClpX protein and downregulation of ribosomal proteins such as RpsT and RpmB are suggested to occur because of the involvement of these OMV genes in ribosomal 30S and 50S synthesis, respectively [ 43 , 44 ].…”