Fig. 6.

Expression of the C. marina FaRel RSH enzyme leads to overproduction of the ppGpp and ppApp alarmones and depletion of intracellular GTP and ATP. (A) Induction of FaRel triggers nucleoid decondensation in E. coli. Depicted are phase-contrast (Upper) and fluorescence images (Upper Middle, Lower Middle, and Lower) of E. coli cells costained with DNA dye DAPI and outer membrane dye FM 5-95. The representative cells carry either an empty or FaRel-expressing vector, and are imaged under uninducing (Mops−glucose medium) or inducing (15 min in Mops−glycerol−arabinose medium) conditions. Note the loss of visible nucleoid structure upon induction of FaRel. As a positive control, cells containing empty vector (Mops−glucose medium) were incubated with rifampicin, which triggers nucleoid decondensation through inhibition of transcription. See SI Appendix, Fig. S10 for a larger field of view with more cells. (B) Pulse-labeling assays following incorporation of ³H-uridine (black traces), ³⁵S-methionine (red traces), and ³H-thymidine (blue traces). E. coli BW25113 cells transformed with empty vector control plasmid pBAD33 were treated with 300 µg/mL kanamycin, 100 µg/mL rifampicin, and 30 µg/mL nalidixic acid as controls for specific inhibition of translation, transcription, and replication, respectively (Left). Expression of FaRel from the pBAD33-faRel plasmid was induced with 0.2% l-arabinose (Right). (C) The expression of C. marina FaRel leads to the accumulation of the alarmone ppGpp as detected by TLC. Alarmone accumulation is efficiently counteracted by wild-type aTfaRel but not its enzymatically compromised D54A mutant. Autoradiograms of a representative TLC plate and a biological replicate (SI Appendix, Fig. S12) are presented. (D and E) Nucleotide pools in E. coli BW25113 expressing C. marina faRel alone. Cell cultures were grown in defined minimal Mops medium supplemented with 0.5% glycerol at 37 °C with vigorous aeration. Expression of C. marina faRel was induced with 0.2% l-arabinose at OD₆₀₀ 0.5. Intracellular nucleotides are expressed in pmol per OD₆₀₀ per milliliter of culture as per the key in each plot. Error bars indicate the SE of the arithmetic mean of three biological replicates. (F) The ³²P-ppApp synthesis assays with either 0.5 µM C. marina faRel Y175A or 30 nM E. coli RelA supplemented with starved ribosomal complexes (0.25 µM 70S IC(MV) + 2 µM tRNA^Val) using 0.5 mM ³²P ATP and 1 mM ADP as substrates. Experiments were performed in Hepes:Polymix buffer, pH 7.5 at 37 °C, in the presence of 5 mM Mg²⁺. (G) Neutralization of C. marina faRel toxicity by overexpression of E. coli SpoT. Expression of SpoT from pMG25 was driven by 50 µM IPTG, and expression of faRel from pBAD33 was driven by 0.2% arabinose.