## 2 Results and Discussion
### Global Overview of the Rsh\({}_{\text{US6-1}}\) Regulon
However, genes in the Rsh\({}_{\text{US6-1}}\) regulon were numerous and the proteins which can modulate the expression of _luxI/R_ homologs vary in bacteria [44]. Therefore, the transcriptome data were used to investigate the potential molecular mechanisms of the decreased expression of _novI_ and _novR_ in _Arsh_. During exponential growth, transcription is widely under control of the housekeeping sigma factor \(\sigma^{70}\). ppGpp can regulate the expression of genes via the regulation of alternative sigma factor competition [45]. Previous studies showed that QS was influenced by alternative sigma factors. In _P. aeruginosa_ PAO1 and _P. fluorescens_ UK4, ppGpp up-regulates the expression of _rpo_S encoding a stationary phase sigma factor and RpoS increases the expression level of AHL-related genes as well as the AHL production [16; 46]. However, the _Pseudomonas_ model might not be applicable in strain US6-1, since its genome does not encode a corresponding RpoS. US6-1 possesses 22 putative sigma \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Genes** & **Strains** & **24 h** & **36 h** & **48 h** & **96 h** \\ \hline _novI_ & US6-1 & 1.166 \(\pm\) 0.773 & 1.060 \(\pm\) 0.466 & 1.115 \(\pm\) 0.647 & 1.039 \(\pm\) 0.356 \\  & \(\Delta\)_rsh_ & 0.261 \(\pm\) 0.083 & 0.011 \(\pm\) 0.002 * & 0.020 \(\pm\) 0.004 * & 0.026 \(\pm\) 0.004 ** \\  & \(\Delta\)_rsh_ (pCM62-rsh) & 0.501 \(\pm\) 0.085 *** & 0.648 \(\pm\) 0.216 & 0.753 \(\pm\) 0.097 & 3.975 \(\pm\) 1.547 * \\ _novR_ & US6-1 & 1.012 \(\pm\) 0.185 & 1.006 \(\pm\) 0.129 & 1.001 \(\pm\) 0.055 & 1.029 \(\pm\) 0.314 \\  & \(\Delta\)_rsh_ & 0.119 \(\pm\) 0.018 ** & 0.068 \(\pm\) 0.005 *** & 0.547 \(\pm\) 0.017 *** & 0.881 \(\pm\) 0.354 \\  & \(\Delta\)_rsh_ (pCM62-rsh) & 0.601 \(\pm\) 0.050 ** & 0.463 \(\pm\) 0.076 & 0.543 \(\pm\) 0.054 & 2.486 \(\pm\) 0.972 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Relative expression levels of _novI_ and _novR_ by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RNA samples were extracted from bacterial cultures after 24 h, 36 h, 48 h and 72 h. The levels of gene expression in US6-1, \(\Delta\)_rsh_, \(\Delta\)_rsh_ (pCM62-rsh) were normalized to 16S rRNA gene, and the relative levels in _Arsh_ and _Arsh_ (pCM62-rsh) to that in the wild-type strain (set as value of 1) were reported. Values shown are the average of biological triplicate experiments with standard deviations (mean \(\pm\) standard deviation (SD)). *** represents \(p\) \(<\) 0.05; *** represents \(p\) \(<\) 0.01; *** represents \(p\) \(<\) 0.001. Figure 5: Identification of degradation of the medium-chain AHL by \(\Delta\)_rsh_. Strain \(\Delta\)_rsh_ was incubated with 2 uM C8-AHL (**a**) and 3-OH-C8-AHL (**b**) in culture medium for 24 h (early exponential growth phase), 48 h (late exponential growth phase) and 72 h (stationary phase), after which the EA extracts were spotted onto AHL reporter plates mixed with A136. The diameters of the blue stains represent the quantity of residual AHL signals. To avoid the influence of the AHL produced by strain US6-1 in the exponential growth phase, _novI_ deletion mutant (\(\Delta\)_novI_) which could not produce AHL was used as positive control. Blank medium (BM) with AHL was used as negative control. factors. Only one housekeeping sigma factor \(\sigma^{70}\) (WP_007014490) was up-regulated 2.2-fold while a extracytoplasmic function (ECF) sigma factor (WP_007013352) was down-regulated 2.73-fold in \(\Delta\)_rsh_. The link between ECF sigma factor and QS has recently given rise to controversy. ECF sigma factors can bind directly to -10 and -35 elements in the _luxR_ promoter, thus inducing _luxR_ expression. The binding sites of this kind of sigma factors are also identified in the upstream region of _luxS_[47; 48]. However, the effect of ECF sigma factors on QS was not observed in another study [49]. Our data showed that _dksA_ (WP_007012876) was up-regulated significantly (6.41-fold) in \(\Delta\)_rsh_.