## 2 Results and Discussion
### Global Overview of the Rsh\({}_{\text{US6-1}}\) Regulon  
The RNA-seq of strain US6-1 and \(\Delta rsh\) cultivated in static conditions, in which US6-1 accumulated AHL signals, was conducted to determine the Rsh\({}_{\text{US6-1}}\) regulon. An average of 24 million clean reads in one sample were obtained. The total mapping radios and uniquely mapping ratios of clean reads to reference genome of US6-1 were up to 98% and 95%, indicating that the sequencing was deep enough to cover almost all kinds of transcripts in the cells (Table S1). The genome of US6-1 contains 5110 annotated protein-encoding genes, 59 pseudo genes and 82 RNA genes [25]. The comparative transcriptome analysis between strain US6-1 and \(\Delta rsh\) revealed 1540 Rsh\({}_{\text{US6-1}}\)-dependent differentially expressed genes (DEGs), which were defined according to the combination of the absolute value  
Figure 3: ppGpp accumulation in strain US6-1, \(\Delta rsh\) and \(\Delta rsh\) (pCM62-rsh) in different growth phases. Levels of ppGpp in cultures of US6-1 (**a**,**d**), \(\Delta rsh\) (**b**,**e**) and \(\Delta rsh\) (pCM62-rsh) (**c**,**f**) in the exponential growth phase (36 h) and in the stationary phase (72 h) were monitored by reverse phase high-performance liquid chromatography (HPLC) analysis. The eluted nucleotides were monitored at 254 nm and identified by comparison with the retention time of 100 uM ppGpp standard. ppGpp standard was eluted at 71 min under the current conditions and the position is indicated by an arrow. of a fold change \(\geq\)2 and an adjust \(p\) value \(\leq\) 0.05 (Table S2). Compared with the wild type, 911 of these genes were up-regulated (17.83%) and 629 genes (12.31%) were down-regulated in \(\Delta\)_rsh_ (\(\Delta\)_rsh_ vs. US6-1). The differential expression varied between an 8.82-fold up-regulation of a gene encoding a protein of ferrisiderophore receptor (WP_007014902) and a 40.5-fold down-regulation of an autoinducer synthesis protein (WP_007013362). The transcriptome data were validated by analyzing the expression levels of 39 representative genes using qRT-PCR (Table S3). The result showed that 29 (74.36%) of the tested genes were regulated in the same direction (up or down) in both RNA-seq data and qRT-PCR (\(\Delta\)_rsh_ vs. US6-1), and the fold change of each gene in qRT-PCR was \(\log_{2}(x)>1\) or \(\log_{2}(1/x)<-1\) (representing a plain fold-change >2) with statistical differences. Among these 29 genes, the expression of 24 genes by qRT-PCR (the complementary strain vs. US6-1) was not different significantly or was regulated in the opposite direction in the RNA-seq data. Therefore, the transcriptome data were in good agreement with the qRT-PCR data, which proved the reliability of the transcriptome data. These genes were further grouped based on Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and pathway enrichment analysis (Figure 4a). The [PERSON]'s exact test identified 12 significantly over-represented KEGG pathways. The result suggested that \(\mathrm{Rsh_{US6-1}}\) controlled a variety of metabolic pathways. The most of the DEGs involved in ribosomal protein production, porphyrin and chlorophyll metabolism, amino acyl-tRNA biosynthesis, oxidative phosphorylation, TCA cycle, phenylalanine, tyrosine and tryptophan biosynthesis were up-regulated in the mutant, which suggested that the role of \(\mathrm{Rsh_{US6-1}}\), similar to that in _E. coli_, was to repress the majority of cellular processes to reallocate cellular resources [39]. Although one of the hallmarks of stringent response is the induction of amino acid biosynthesis [39; 40], unexpectedly, in \(\Delta\)_rsh_ most genes involved in amino acid biosynthesis were up-regulated. Similarly, in _relA_ mutant of _Pectobacterium atroseticum_, several unlinked clusters of genes involved in branched chain amino acid metabolism (_ilvGMEDA_, _ilvIH_, _ilvBN_ and _leuABCD_) were also up-regulated [41]. There may be other mechanisms which can induce amino acid biosynthesis when US6-1 grows under environmental stress.