Thus, depletion

of YgjD protein leads to a pool of un- or undermodified transfer-RNAs (as described by [8]), possibly resulting in non-optimal interactions between transfer-RNAs and mRNA inside the ribosome. This could potentially elicit a stringent-response like program (governed by (p)ppGpp release) and explain the phenotypic consequences check details of YgjD depletion that we observed. Non-optimal interactions between non-modified tRNAs and mRNA could be similar to the effects caused by ribosomes that are stalled on “”hungry”" codons: these codons are unsuccessfully trying to pair with either rare transfer-RNAs or transfer-RNAs that are non-aminoacylated due to amino-acid limitation. Hungry codons can provoke the production of aberrant proteins by frame shifts, slides of the translational machinery or incorporation of noncognate transfer-RNAs [34, 35]. This might also explain the slow onset of the consequences of YgjD depletion: accumulation of aberrant proteins would slowly increase over time and reach a level where Doxorubicin supplier several cellular processes might be affected simultaneously. Although the biochemical activity of YgjD has been described [8], the cellular functions of YgjD are not completely resolved. It

will be interesting to ask how the proteins in the YgjD/YeaZ/YjeE complex [3] of Escherichia coli are interacting to fulfill their functions, and to ask whether YgjD is involved in other cellular processes or responding to environmental cues. Single-cell observations of YgjD depletion experiments might be helpful to generate and test hypotheses about the essential role of this protein, and to help explain why it is so widely conserved. Methods Bacterial strains and growth medium P1 transduction and TSS transformation were performed as described elsewhere [36, 37]. Strain DY330 as well as strains harboring the plasmid pCP20 [38] were grown at 32°. All other strains were grown at 37°. To grow

TB80 and TB84 under permissive conditions, we used LB medium (Sigma) supplemented with 0.1% (batch culture) or 0.01% (before time-lapse microscopy) L-arabinose (Sigma). LB ever agar (1.5% agar) was from Sigma, and used for preparing agar plates and agar pads for time-lapse microscopy. Strain construction Strains containing more than one knockout or marker were generated by sequential P1-transductions. Resistance markers were removed by Flp recombinase mediated site-specific recombination [39]. To control expression of ygjD, we constructed a conditional mutant with a second copy of the promoter of the araBAD operon in front of the native chromosomal locus of ygjD by directly inserting a Para-construct in front of ygjD, as described previously [40]. Removal of L-arabinose and addition of glucose allows tight repression of target genes under control of Para [40, 41].