## 2 Results and Discussion
### Rsh\({}_{\text{USS6-1}}\) Regulon Involved in Other Processes  
The large plasmids present in US6-1 possess putative biodegradation genes that play a key role in PAH degradation [56]. These genes were investigated in the transcriptome data. Two genes (WP_007014585, WP_007014540) located in the large plasmid pLA3 showed up-regulation in \(\Delta\)_rsh_, which was similar to the situation in _Sphingomonas_ sp. LH128 [14]. For strain LH128, the expression of phenanthrene catabolic genes were decreased while _rsh_ was up-regulated after 6 months of starvation. This indicated that Rsh\({}_{\text{US6-1}}\) might affect the degradation of PAH. Many researches on sphingomonads mainly focused on enzymes which could degrade PAH directly [56]. Considering sphingomonads always degrade aromatic compounds in contaminated soil with limited nutrient availability [7], studying the relationship between Rsh and genes involved in the degradation of aromatic compounds would help understand a comprehensive degradation mechanism. Future studies carried out in an environment contaminated with PAH using \(\Delta\)_rsh_ are needed to investigate the relationship. All sphingomonads contain in their outer membranes sphingolipids which replace lipopolysaccharideaccharides [7]. Serine palmitosyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis [57]. Two up-regulated genes (WP_007012867 and WP_007014840) were annotated as \(\alpha\)-oxoamine synthases, a family of enzymes including SPT. Their identification of amino acid sequences with SPT from _Sphingomonas paucimbilis_ and _Sphingomonas wittichii_ were more than 30% [57; 58], indicating they were responsible for encoding SPT. Sphingolipids play an important role in bacterial survival under stress [59]. However, our results indicated that when spingomonads encountered environmental stress, sphingolipid biosynthesis was repressed via stringent response instead of being induced, probably for energy and resource saving. Figure 6: Assessment of EPS production of strain US6-1, \(\Delta\)_rsh_ and \(\Delta\)_rsh_ (pCM62-rsh). (**a**) Colony biofilm morphology of strains on the P5Y3 plates containing 40 \(\mathrm{\SIUnitSymbolMicro g}\)/mL Congo red. One representative experiment out of three independent biological replicates is shown. The white bar represents 5 mm. (**b**) Congo red binding assay. The biomass of strains grown in static culture for 96 h was determined gravimetrically as cell wet weigh. The bacterial mass was then mixed with 1 mL of Congo red (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) in 0.9% saline for binding. After removing the biomass, the OD\({}_{\text{490}}\) of unbound Congo red in supernatant was measured and the percentage of Congo red left in supernatant g\({}^{-1}\) was shown. The OD\({}_{\text{490}}\) of 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL Congo red in 0.9% saline was used as the control. The results are averages of three replicates, and the error bars indicate standard deviations. “***” represents \(p\) < 0.001. The expression level of shape determination genes (_rodA_, _mreC_ and _mreB_), cell-division genes (_ftsW_, _ftsQ_) and genes encoding penicillin-binding proteins increased as a result of the deletion of _rsh_US6-1. These genes were involved in cell size and shape, indicating that the morphology of US6-1 was regulated by RshUS6-1. Starvation stress can result in the changes of cell morphology of _Sphingomonas_[14; 60]. The reason might be that the change of cell size and shape improves the uptake of scarce nutrients due to the increase in cell surface area to volume ratio [61]. Our data suggested that US6-1 might adapt to environment stress via the change in cell size or shape regulated by RshUS6-1. Genes responsible for putative regulatory functions were investigated. Several transcriptional regulators including the cold shock protein, an UspA and a PadR family transcriptional regulator were down-regulated.