# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 4 Discussion
The lack of FIS in stationary phase cells might account for _lns_ expression patterns of wt and _relA/spoT_ strains that become similar as the cell density increases (Abs\({}_{600\text{ nm}}>1.5\), Figure 2D). The fact is that (p)ppGpp represses both _fis_ and _lns_ promoters, thereby allowing us to speculate on the occurrence that the _lns_ gene is subjected to a dual negative action by (p)ppGpp: (i) by directly impacting the intrinsic activity of _lns_ promoter; (ii) by depleting the _lns_ gene of its natural activator FIS. Importantly, an adaptative stress-response also takes place when pathogenic bacteria face up transitions from free-living to host-associated states. In this contest, H-NS has been found in important human pathogens such as _S. typhimurium, S. flexneri, V. cholerae, Y. pesti, P. multocida_ and, consistently, many genes controlled by this protein turn out to be virulence genes or genes expressing potential pathogenicity determinants [46; 47; 48; 49; 50; 51]. The response of _lns_ promoter to (p)ppGpp, investigated through an in vitro transcription system (Figure 4), undeniably indicates that the inhibition exerted by this modified nucleotide is direct and promoter specific. In fact, a 50% loss of promoter activity is observed at \(\sim\)400 \(\upmu\)M of (p)ppGpp and the _lns_ transcription declines as much as 5-10% for higher (p)ppGpp concentrations. These amounts of (p)ppGpp are very close to those used in other in vitro studies [52; 11; 53] and consistent with the value (\(\sim\)0.9-1 mM) measured in vivo during the stringent response [42; 54]. The failure to identify _lns_ as a (p)ppGpp-responsive gene using transcriptomics techniques as RNA-seq [11] and microarrays [12] was quite unexpected. Possible explanations for conflicting results are reported below. As already stated, [PERSON] et al. [11] estimated the _lns_ mRNA level at 5 and 10 min after the production of (p)ppGpp by an inducible _relA_ plasmid system. Using an analogous experimental approach (Figure 3A), we show that at least 20-25 min is required to detect significant differences in _lns_ expression between induced and not-induced cells. Concerning the study by [PERSON] et al. [12], the total RNA for DNA chip and Reverse Transcriptase PCR experiments was extracted from cells in stationary phase (Abs\({}_{600}=1.5\)). Under our experimental conditions (Figure 2B,D), at cell density ranging from Abs\({}_{600}=1.5\) to Abs\({}_{600}=3.0\), the level of _lns_ transcript of wt strain is comparable with that of _relA/spoT_ double mutant, whereas a differential expression is observed mainly in exponential phase of growth. Thus, because of narrow time window or cell status, the in vivo (p)ppGpp-mediated regulation of _lns_ might have been overlooked in previous studies [11; 12]. Our results suggest that stationary phase cells slow down the _lns_ transcription rate in a (p)ppGpp-independent manner probably because, at this time, other factors (i.e., the lack of FIS) may contribute mostly to the regulation of this gene. Remarkably, H-NS is also able to bind and repress transcription from its own promoter [15] and it was found to accumulate when cells enter the stationary phase [55]. However, the intracellular levels of this protein during growth-curve still remain a matter of debate [56; 57; 58]. Thus, an increase in H-NS level would enhance the autoregression providing an explanation for the decline of _lns_ mRNA in both wt and _relA/spoT_ strains, as observed at the onset of stationary phase (Abs\({}_{600}\) = 1.5 in Figure 2D). Ultimately, stationary cells (as the starved ones), characterized by a low growth rate and a reduced DNA synthesis need a poor demand for de novo production of the nucleoid protein H-NS. Recently, a large collection of (p)ppGpp directly regulated genes has been obtained thanks to an extensive in vitro transcription analysis carried out on more than 100 promoters (not including _lns_) whose in vivo expression was affected at least twofold by the modified nucleotide [11].