## 2 Results and Discussion
### Global Overview of the Rsh\({}_{\text{US6-1}}\) Regulon
According to the validation of the transcriptome data, DEGs involved in AHL production, sigma factor synthesis, RNA degradation, EPS biosynthesis, PAH degradation, sphingolipid production, cell division and shape and GTP synthesis were chosen, and their expression profiles were visualized in the heat map (Figure 4b, Table S3). Except the genes related to AHL production, the most of these genes were under negative control of \(\mathrm{Rsh_{US6-1}}\). These DEGs will be discussed in detail in the following paragraphs. Figure 4: _Cont._  
Essential Genes for AHL Production were Positively Regulated by Rsh\({}_{\text{U}\text{S}6-1}\) in the Exponential Growth Phase  
There are only one gene _novR_, encoding an autoinducer synthase (WP_007013362) and one gene _novR_ encoding a LuxR family transcriptional regulator containing an autoinducer binding domain (WP_007013363) in the genome of US6-1. These two genes are essential for AHL production. The transcriptome data showed that in strain \(\Delta\)_rsh_, _novI_ and _novR_ were down-regulated 40.5 and 2.35-fold at 36 h of incubation, respectively. The qRT-PCR analysis of culture samples from different growth periods confirmed the decreased expression levels of _novI_ and _novR_ in \(\Delta\)_rsh_ (Table 1). The _novI_ and _novR_ were both down-regulated significantly at 36 h and 48 h (exponential growth phase) in \(\Delta\)_rsh_ while \(\Delta\)_rsh_ (pCM62-rsh) could complemented their expression. Although the essential genes for AHL production were down-regulated in the exponential growth phase due to the lack of ppGpp, \(\Delta\)_rsh_ still possibly produced low concentrations of AHL signals. Since US6-1 could degrade AHL signals, we analyzed whether \(\Delta\)_rsh_ degraded AHL in the exponential growth phase. When provided with 2 uM medium-chain AHL (C8-AHL and 3-OH-C8-AHL) as cosubstrates in culture medium, \(\Delta\)_rsh_ did not show the capacity to degrade the medium-chain AHL at 48 h (Figure 5). However, at 72 h of incubation, there were fewer AHL signals in \(\Delta\)_rsh_ cultures. These results suggested that \(\Delta\)_rsh_ might not degrade AHL signals in the exponential growth phase. The activation of its capacity to degrade AHL was likely related to the entrance to the stationary phase. Overall, the reason why Rsh\({}_{\text{U}\text{S}6-1}\) was required for the AHL accumulation was that Rsh\({}_{\text{U}\text{S}6-1}\) positively regulated essential genes for AHL production in the exponential growth phase, and might be unrelated to the AHL degradation. Figure 4: Differentially expressed genes (DEGs) overview (\(\Delta\)_rsh_ vs. US6-1) in the RNA-seq data. (**a**) Over-represented Kyoto Encyclopedia of Genes and Genomes (KEGG) categories. KEGG annotation and pathway enrichment analysis was performed using the KEGG pathway database ([[http://www.genome.jp/kegg/](http://www.genome.jp/kegg/)]([http://www.genome.jp/kegg/](http://www.genome.jp/kegg/))). The \(p\) values denote the enriched levels in a KEGG pathway, which was calculated using a [PERSON]’s exact test [42]. Up- and down-regulated genes (\(\Delta\)_rsh_ vs. US6-1) are represented by red and blue bars respectively, representing the number of genes per functional category. (**b**) Heat map of log\({}_{2}\) expression ratios of specific DEGs involved in AHL production, sigma factor synthesis, RNA degradation, EPS biosynthesis, PAH degradation, sphingolipid production, cell division and shape, GTP synthesis and other functions in strain US6-1 and \(\Delta\)_rsh_. Expression values are reflected by red-blue coloring as indicated. The heat map was drawn by the software Heml [43]. Potential Molecular Mechanisms of Rsh\({}_{\text{US6-1}}\)-Dependent Expression of novel and nov\(R\)  
In the case of _E. coli_, (p)ppGpp regulates the expression of genes through binding to \(\beta\)'-subunit of RNA polymerase to destabilize the short-lived open complexes that form at certain promoters [3].