# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 3 Results  
### Low Levels of (p)ppGpp Stimulate lns Expression  
The role of (p)ppGpp on _lns_ expression was initially investigated by means of antibiotics as chloramphenicol (CAM) and kanamycin (Kan), which were known to affect the intracellular level of this unusual nucleotide. In fact, these two drugs, by blocking protein synthesis, have been shown to cause a dramatic reduction in (p)ppGpp [41]. Thus, to verify whether (p)ppGpp could regulate _lns_ expression, the level of _lns_ mRNA was monitored in a time course experiment after treating the cell with high doses of CAM and Kan. As seen in Figure 1, both drugs behave similarly by triggering a prompt increase (\(\sim\)threefold) of _lns_ transcript compared to the untreated cells as control. Steady-state level of _lns_ mRNA was further investigated using a strain defective for ReIA synthetase, the enzyme catalyzing (p)ppGpp production along with double mutant _relA/spoT_ that, in addition to _relA_, carried an inactivated bifunctional (p)ppGpp synthetase/hydrolase SpoT. [PERSON] et al. [33] found that glucose exhaustion during cell growth provoked (p)ppGpp accumulation. Thus, the level of this effector progressively diminished passing from wild type (wt) to _relA_ and from _relA_ to _relA/spoT_ backgrounds. Accordingly, northern analysis demonstrates that basal level of _lns_ transcript (Figure 2A, time 0), shows \(\sim\) three- and sevenfold increase in _relA_ and _relA/spoT_ mutants, respectively, compared to wt strain. In addition, chloramphenicol treatment induces a sudden burst of _lns_ expression in all the strains considered, even if at different extents with respect to the intrinsically different constitutive levels of _lns_ mRNA (Figure 2A,C). Indeed, the lower the basal steady-state level, the greater is the induction of _lns_ transcription caused by the CAM-dependent reduction in (p)ppGpp. Thus, this stimulation decreases from wt (fivefold) to _relA/spoT_ (1.4-fold) through _relA_ (twofold) cells; given that the intracellular level of (p)ppGpp is already very low in _relA/spoT_ strain, CAM effects are almost completely lost in this double mutant (Figure 2C). To reinforce this finding, the level of _lns_ transcript was measured during cell growth in wt and _relA/spoT_ strains. Northern analysis reveals that _lns_ expression is \(\sim\) twofold higher in double mutant compared to wt cells in the exponential phase of growth (A\({}_{600}<1\)) and this difference gradually disappears as the cells approach the stationary phase (A\({}_{600}>1.5\)) (Figure 2B,D). Taken together, these results strongly suggest that _lns_ expression is inversely correlated with the intracellular concentration of (p)ppGpp. Figure 1: Effects of chloramphenicol and kanamycin on _lns_ transcription in vivo. The _E. coli_ strain MRE600 was grown at 37 \({}^{\circ}\)C in LB broth up to Abs\({}_{600}\) = 0.5–0.6 (exponential phase of growth) and at the indicated times after the addition of 200 ug/mL of chloramphenicol (**A**) or 300 ug/mL of kanamycin (**B**), cell aliquots were harvested for RNA extraction. About 10 ug of total RNA were subjected to northern blot analysis and membranes hybridized with a [\({}^{32}\)P]-labeled _lms_ probe. Treated cells (\(\bullet\)) and control culture (C) in the absence of drugs (\(\blacktriangle\)) are shown. Data represent the average of at least two independent experiments and standard deviation is reported. Figure 2: Chloramphenicol and mutations of _relA_ and _spoT_ genes induce _lns_ transcription in vivo. The _E. coli_ wt strain CF2676 (\(\bullet\)) and its _relA_ (\(\blacksquare\)) and _relA_/_spoT_ (\(\blacktriangle\)) derivatives were grown at 37 \({}^{\circ}\)C in LB broth up to the exponential phase (Abs\({}_{600}\) = 0.5–0.6). Then the steady-state level of _lns_ mRNA was monitored by northern analysis at the indicated times after addition of chloramphenicol (200 ug/mL) (**A**) and as a function of the cell density (Abs\({}_{600}\)) (**B**).