# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 1 Introduction  
The signaling molecules guanosine-tetraphosphate (ppGpp) and guanosine-pentaphosphate (ppGpp), collectively named (p)ppGpp, are the main mediators of \"stringent response,\" a common phenomenon occurring when bacteria face environmental stresses [1; 2; 3]. Two enzymes are responsible for the production of (p)ppGpp, the major ReLA synthase, and the SpoT degradase that displays both (p)ppGpp degradation and synthetic activity [4]. In particular, when bound to ribosome, the ReLA enzyme senses through its autoinhibitory domain and is being activated by the entrance of uncharged tRNAs into the ribosomal A-site, a circumstance occurring during amino acid starvation [5]. Thus, nutrients limitation causes an intracellular accumulation of (p)ppGpp that dramatically alters bacterial physiology and metabolism [6]. Under this condition, indeed, growth rate is slowed, stable RNA (rRNA and tRNA) production is inhibited, and the high levels of modified nucleotide differentially up- or down-regulate the expression of a large number of genes. The stringent response has been investigated for half a century, and solid evidence for (p)ppGpp acting directly on RNA polymerase (RNAP) has been already reported [7; 8]. However, only two recent studies have provided deeper insights into the mechanism of action exerted by (p)ppGpp in modulation of transcription initiation from specific promoters; (p)ppGpp was found to target the _E. coli_ RNAP and two binding sites for this alarmone have been localized on the core enzyme [9; 10]. In fact, the first site is at the interface of \(\beta\)and \(\omega\) subunits of RNAP, while the second one originates from the contact point of the transcription factor DksA with RNAP. Recently, by means of high-throughput sequencing technologies (RNA-seq), a complete collection of _E. coli_ promoters, controlled in vivo by (p)ppGpp, has been provided [11]. This study shows that 757 genes are up- or down-regulated (at least twofold) within five minutes of continuous production of (p)ppGpp through the conditional expression of a mutated RelA lacking its autoinhibitory domain. Even if [PERSON] et al. [11] widened the list of (p)ppGpp-responding promoters (~75% were not previously identified), under the experimental conditions used, the _lns_ gene encoding the Histone-like Nucleoid Structuring (H-NS) protein was not identified. Similarly, also [PERSON] et al. [12] did not find any (p)ppGpp-dependent changes of _lns_ expression by a microarray analysis. The H-NS protein has been extensively investigated in our laboratory, and its biochemical characterization and the regulation of this gene date back to the late 1980s [13; 14; 15]. Currently, H-NS, in addition to its well-established role in the organization of bacterial chromatin, is recognized as a \"universal repressor\" involved directly or indirectly in the regulation of numerous genes (~200), most of them linked to stress responses and bacterial pathogenesis [16; 17; 18; 19]. The basis for H-NS function as a gene silencer resides in its preferential binding to AT-rich curved DNA sequences [20; 21], often found upstream of _E. coli_ promoters and in its ability to induce bending of noncurved DNA [22], thus altering DNA conformation/topology and/or competing with RNAP and other regulators [23; 24; 25; 26]. Genome-wide functional analyses have mapped ~1000 sites bound by H-NS, accounting for the regulation of 5% of the entire _E. coli_ genome [27], including DNA sequences of exogenous origin acquired by horizontal gene transfer [28; 29]. In a previous study [30], we have found that the activation of _lns_, upon cell resumption from stationary phase, is partially abolished by high levels of (p)ppGpp. Here, we have provided a deeper investigation on the role of (p)ppGpp in the regulation of _lns_, showing that in vivo level of _lns_ mRNA inversely correlates with the intracellular concentration of the modified nucleotide.