Genetic interaction screens with ordered overexpression and deletion clone sets implicate the Escherichia coli GTPase YjeQ in late ribosome biogenesis

Abstract

The Escherichia coli protein YjeQ is a circularly permuted GTPase that is broadly conserved in bacteria. An emerging body of evidence, including cofractionation and in vitro binding to the ribosome, altered polysome profiles after YjeQ depletion, and stimulation of GTPase activity by ribosomes, suggests that YjeQ is involved in ribosome function. The growth of strains lacking YjeQ in culture is severely compromised. Here, we probed the cellular function of YjeQ with genetic screens of ordered E. coli genomic libraries for suppressors and enhancers of the slow-growth phenotype of a delta yjeQ strain. Screening for suppressors using an ordered library of 374 clones overexpressing essential genes and genes associated with ribosome function revealed that two GTPases, Era and initiation factor 2, ameliorated the growth and polysome defects of the delta yjeQ strain. In addition, seven bona fide enhancers of slow growth were identified (delta tgt, delta ksgA, delta ssrA, delta rimM, delta rluD, delta trmE/mnmE, and delta trmU/mnmA) among 39 deletions (in genes associated with ribosome function) that we constructed in the delta yjeQ genetic background. Taken in context, our work is most consistent with the hypothesis that YjeQ has a role in late 30S subunit biogenesis.

FIG. 1.

Growth phenotype and suppressor screening for correction of the slow growth of the ΔyjeQ strain. (A) Growth curves for the ΔyjeQ strain and wild-type E. coli. Symbols: •, wild type plus pCA24N-yjeQ; ○, wild type plus pCA24N; ▾, ΔyjeQ plus pCA24N-yjeQ; ▿, ΔyjeQ plus pCA24N. (B) Primary screen for suppressors of ΔyjeQ slow growth. The time point used correlates to 340 min in panel A. The filled circles show the results for the high-copy-number control (ΔyjeQ plus pCA24N-yjeQ), and the open circles show the growth data for each individual clone screened. The solid line indicates the mean of the high-copy-number control, the dashed line indicates the sample mean, and the dotted line indicates 1.5 standard deviations above the sample mean.

FIG. 2.

Ribosome profiles for wild-type and ΔyjeQ E. coli. The strains used are indicated. The profiles were prepared using analytical ultracentrifugation. WT, wild type.

FIG. 3.

Suppression of the ΔyjeQ slow growth by correction of the defective ribosome profile. In the growth curves circles indicate wild-type E. coli and inverted triangles indicate ΔyjeQ E. coli; filled symbols indicate that the pCA24N plasmid with the gene indicated was overexpressed, and open symbols indicate that pCA24N was is present. Ribosome profiles were prepared using analytical ultracentrifugation. (A) Suppression by overexpression of infB. (B) Suppression by overexpression of era.

FIG. 4.

Ribosome profiles of double-deletion strains. Each double-deletion ribosome profile resembles the ribosome profile of the parent single-deletion strain. The strains used are indicated. The ribosome profiles were prepared using analytical ultracentrifugation.

FIG. 5.

Genetic interaction network for yjeQ. Genes interacting with yjeQ on the left are multicopy suppressors of the yjeQ deletion mutant slow growth. Deletions of the genes interacting with yjeQ on the right are slow-growth enhancers. The overlap between the two gene sets is shown in the center. Gene functions are color coded; blue indicates genes that code for proteins involved in translation, green indicates genes that encode ribosome biogenesis factors, orange indicates genes that are general metabolism genes, and gray indicates a gene that encodes a protein with an unknown function.