Overexpression of YbeY partially rescues the ΔybeX phenotype
We used a constitutively expressed E. coli open reading frame (ORF) plasmid library representing 3974 ORFs, cloned in a multi-copy zeocin-resistant Gateway™ pENTR/Zeo plasmid (Rajagopala et al. , 2010), to search for genes that compensate for the deletion ofybeX . The selection was made in two independent experiments for three 12-hour rounds, resulting in enriched plasmid pools (Fig. 9A ). We sequenced about 150 clones from the enriched plasmid pools. TheybeY -coding plasmid was predominant (>90%) among sequenced clones. Surprisingly, we did not recover a single ybeXcoding plasmid, suggesting the harmfulness of ybeXoverexpression. Accordingly, the multi-copy ybeX -coding plasmid led to equally strong growth inhibition in WT and ΔybeX cells (Fig. S8a ). In contrast, the ybeY -coding plasmid had no visible detrimental effect on the growth of the ΔybeX mutant.
We also measured growth in liquid LB medium. In this experiment, the multi-copy ybeX plasmid in the ΔybeX background further increased the duration of the lag phase while also leading to a lower plateau of the growth curve (Fig. S8b ). In contrast, the multi-copy ybeY plasmid in the ΔybeX background partially rescues the lag phase phenotype (Fig. S8b ). In the light of this partial rescue of the ΔybeX phenotype by the multi-copyybeY coding plasmid, we next tested whether overexpression of YbeY from an inducible pET-based multi-copy plasmid under the control of the taq promoter can further rescue the ΔybeX lag phenotype. We found that overexpression of YbeY in ΔybeX strain in the presence of 1 mM IPTG did not result in complete rescue of the ΔybeX lag phase phenotype (Fig. 9B ).
We further tested whether overexpression of YbeX and YbeY from a single-copy TransBac library plasmid influences the growth ofΔybeY phenotype (Fig. 9C ). Overexpression of YbeX and YbeY conferred no growth effect on WT cells. When induced inΔybeY cells, the single-copy ybeY plasmid compensated for the lack of chromosomal ybeY . In contrast, overexpression of YbeX in ΔybeY cells led to a strong growth-rate reduction and a markedly lower final culture density.
Further, we conjugated the TransBac library plasmid overexpressing YbeX into three additional Keio deletion strains of genes whose products are associated with 30S ribosomal assembly. Deletion of rimM andyjeQ is known to impede growth at 37°C, while ksgAdeletion does not affect bacterial growth at 37°C (Shajani et al. , 2011). We found that the growth of ΔksgA was not significantly affected by YbeX overexpression, while ΔrimM andΔyjeQ strains exhibited growth inhibition (Fig. 9D ,E ). Therefore, sensitivity to ybeX overexpression is not a YbeY-specific phenomenon but seems to be associated with defective ribosomal assembly in general.