# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 4 Discussion  
Bacterial cells, lacking homeostatic mechanisms, withstand changes in environmental conditions, deeply reshaping transcriptional activity. Hence, prompt adaptation to stresses, in particular to amino acid starvation, is coupled with an intracellular accumulation of the second messenger (p)ppGpp, also known as \"magic spot\". This signaling molecule modulates the stringent response by affecting several fundamental cellular processes, including transcription. In this study, we demonstrate that the expression of _lms_ gene, coding for the DNA-binding protein H-NS, is under control of (p)ppGpp. Specifically, treatments with antibiotics capable of arresting the  
Figure 4: (p)ppGpp represses the in vitro transcription of _lms_. The activity of _lms_ promoter was investigated by an in vitro assay as a function of increasing concentrations of (p)ppGpp. The supercoiled plasmid pPK400::_cat_ (-100 ng) used as DNA template was incubated for 10 min at 37 \({}^{\circ}\)C with the _E. coli_ RNA polymerase and the indicated amounts of (p)ppGpp. Then, transcription was stopped, and RNA analyzed by northern blot hybridized with a [32P]-labeled _cat_ probe obtained from pCM7 (Pharmacia) (**A**). Asterisks indicate the two _cat_ mRNAs that start from the _lms_ promoter and end at the _rmB_ ribosomal RNA T1 or T2 terminators located downstream _cat_ gene on pKK400. Transcription is expressed as percentage (%) taking the mRNA level in absence of (p)ppGpp as 100% (**B**). Reactions and processing of samples are described in detail in Materials and Methods. A representative gel is shown, and data correspond to the average of two independent experiments with duplicated points and standard deviation. Figure 5: (p)ppGpp destabilizes the RNAP-_lms_ promoter complex. EMSA was carried out as described in Materials and Methods by incubating for the indicated times, a [32P]-labeled DNA fragment (221 bp), corresponding to _lms_ promoter with RNA polymerase in the absence or presence of ppGpp (500 \(\upmu\)M) (**A**). The electrophoretic migrations of free DNA and _lms_ DNA-RNAP complex are indicated and C is the control sample in absence of protein. Radioactivity associated to free and bound DNA was quantified and complexed DNA has been expressed as percentage (**B**). A representative gel is shown, and the average of four independent experiments and standard deviation are reported. protein synthesis and null mutations in _relA_ and _spoT_ genes responsible for (p)ppGpp production reduce the alarmome level and as consequence elicit an activation of _lns_ promoter in vivo (Figures 1 and 2). Conversely, artificially increasing (p)ppGpp by a recombinant RelA-based system, causes a severe inhibition of the activity of _lns_ promoter (Figure 3). According to these findings and since (p)ppGpp dramatically reprograms the entire transcription profile of bacterial cell, the fact that a regulatory gene as _lns_ is a preferential target of this alarmome is not surprising, and may reflect a valid strategy to enhance the regulative ability of (p)ppGpp itself. In fact, H-NS, besides its role in DNA-packaging and nucleoid organization, shows high pleiotropic effects, and is currently considered as a global repressor of transcription in bacteria. The majority of genes belonging to H-NS regulon are involved in counteracting adverse growth conditions, and in particular H-NS is able to sense shifts of temperature, osmolarity, and pH [44]. Thus, (p)ppGpp, making H-NS decrease below a certain threshold level, might, in turn, determine a derepression of all the genes, commonly silenced by H-NS, which are essential to counteracting stress situation. This assumption is supported by the observation that other nucleoid-associated proteins, mostly those with pronounced regulatory functions such as FIS, Dps, DnaA, StpA, and Hfq, are selectively up- or down-regulated by (p)ppGpp [11]. In particular, FIS (Factor for Inversion Stimulation) was found to stimulate _lns_ expression limited to the exponential phase of growth [45].