# Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in _E. coli_
## 1 Introduction  
Most of the bacterial population resides in a non-proliferating growth state termed the stationary phase. One of the most common environmental signals that prompt bacteria into adopting a non-proliferative behavior is nutrient deprivation [1]. In fact, environmental conditions that would allow constant cell division and bacterial growth are rarely encountered in nature [2]. Stationary phase bacterial cultures are not homogenous but represent a mixture of actively dividing and dying cells [1]. It is also during this phase that a minor population of dormant and non-dividing bacteria, termed persists, arises [3, 4, 5]. Even though persister cells are already detectable in exponential phase cultures, their abundance increases upon entry into the stationary phase [1]. Due to their metabolic inactivity, persister cells exhibit an antibiotic tolerance phenotype [6, 7] that poses a severe health threat and has been linked to recurrent bacterial infections. Pathogenic bacteria within the mammalian host often resort to dormancy to bypass immune response and cope with nutritive stress [5]. Therefore, entry into and exit from the stationary phase are crucial for the long-term survival of a bacterial population and are, therefore, tightly regulated processes. A key player in this crucial transition in Gram-negative bacteria is the alternative sigma factor RpoS, whose expression peaks in the stationary phase [8]. RpoS orchestrates the global downregulation of gene expression in favor of the upregulation of a subset ofgenes critical for survival under these challenging conditions. Furthermore, RpoS has been reported to aid in persister cell formation [9; 10] and appears to promote dormancy hand in hand with (p)ppGpp [11], the alarmone regulator of the stringent response in bacteria [12]. Interestingly, RpoS expression is under the control of several sRNAs, both positively and negatively [13]. As such, the bacterial stress response and stationary phase behavior appear to be regulated in a hierarchal fashion, often relying on small ncRNA regulators. In bacteria, the discovery of sRNA regulators has preceded that of their counterparts in eukaryotes, and these ribo-regulators can be divided into several categories based on their nature, their various modes of action, and the physiological processes that they regulate [14; 15; 16]. Transcriptome profiles unraveled a whole depository of uncategorized sRNAs [17; 18], whose expression levels are heavily affected by key environmental signals. These profiles are significantly dominated by short, trans-encoded sRNAs that regulate gene expression at the post-transcriptional level. A single sRNA can regulate the fate of several target mRNAs via partial base pairing, thereby fine-tuning pathways involved in metabolism [19], quorum sensing [20], antibiotic resistance [21], stress response [22], or virulence [23]. For instance, _ryhB_ appears to aid persister cell formation in uropathogenic _E. coli_[24], while this sRNA has been previously reported to regulate iron metabolism [25]. This further illustrates the intricate nature of RNA regulatory networks in bacteria. These regulatory sRNAs heavily rely on the presence of RNA-binding proteins that protect them against degradation and/or assist them during mRNA target recognition. Hfq, and recently ProQ and CsrA, have been extensively characterized as key RNA chaperones binding, assisting, and stabilizing sRNAs [26; 27; 28]. As such, the identification and characterization of novel sRNAs frequently utilized these chaperones as baits in pull-down studies [29; 30; 31]. Nonetheless, several sRNA could be acting independently of these so far characterized chaperones and would thus be undetected in these pull-down approaches. A so far largely unnoticed class of regulatory RNAs that has recently emerged in transcriptome studies is rancRNAs (ribosome-associated noncoding RNAs).