Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

The uniqueEscherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria,Klebsiella pneumoniae andSalmonella enterica serovar Typhimurium; two pathogenic bacteria,Staphylococcus aureus andNeisseria gonorrhoeae ; and the extremophileDeinococcus radiodurans and then evaluated whether they could functionally complement theE. coli der -null phenotype. OnlyK. pneumoniae andS . Typhimurium Der proteins enabled theE. coli der -null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression ofK. pneumoniae andS . Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused byE. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of theder -null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit.IMPORTANCE InEscherichia coli , Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them,Klebsiella pneumoniae andSalmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly inE. coli . Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation inE. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits.