Characterization Of Quorum Sensing In Pseudoalteromonas Sp. T1lg65 And Its Regulation By Stringent Starvation Protein A

Quorum sensing?QS?is a way for bacteria to socialize by special chemical signal molecules.The signal molecules act as a mediator for communication between bacteria.Bacteria rely on cell density to regulate their own physiological and metabolic activities.Pseudoalteromonas is a new class of marine bacteria,which is widely present in the marine environment and can produce a variety of active substances in Pseudoalteromonas.Diverse studies have found that the production of active substances is regulated by QS,but the research of QS system in Pseudoalteromonas and its regulation mechanism is still lacking.The Pseudoalteromonas sp.T1lg65 with QS intensity was isolated from the Mangrove forest sediments in Ximen Island of Zhejiang Province.The purpose of this paper is to characterize the Lux I/R type QS system characteristics of T1lg65 strain,and to explore the regulation effect of stringent starvation protein A?SspA?on it.According to the genome annotation information,it was found that T1lg65 strain had a signal molecule synthetase-encoded lux I gene and a signal molecule receptor protein-encoded lux R gene.The markerless gene deletion and complement experiments indicated that:?lux I and?lux R lost the ability to produce signal molecules,while?lux I^cand?lux R^crestored the wild-type phenotype,generating blue coloration of indicator A.tumefaciens A136.While expression of the lux I gene in E.coli BL21 strain gained the ability to generate a blue coloration of strain A136.Further analysis of the genome revealed that lux I and lux R are in the upstream and downstream relationship.We found that lux I and lux R are in co-transcription based on PCR detection.In addition,the transcription of lux R was significantly reduced in the?lux I strain,while the transcription of lux I was down-regulated in the?lux R strain.Sequence analysis showed that there may be three“lux-box”sites near the upstream of lux I gene,which are the Lux R binding DNA sites.In summary,the T1lg65 strain has a typical Lux I/Lux R type QS system,and Lux I and Lux R have an interaction relationship.Then,the indicator plate and Thin-layer chromatography?TLC?were used to analyze the signal molecule production and the type of signal molecule.Analysis showed that T1lg65 strain produced the most of signal molecules after shaking for 12 h.It can produce 3-oxo-C₆-HSL,3OH-C₈-HSL,3-oxo-C₈-HSL and C₈-HSL.By exploring the effect of the SspA-HNS-RpoS regulatory system on QS,cross-feeding bioassay results showed that the?rpo S and?hns strains still have the ability to produce signal molecules,but?rpo S strain weakened the indicator A136's ability to show blue,indicating that this strain produces fewer signal molecules.Interestingly,?ssp A strain failed to induce a blue coloration of A136,and the?ssp A^c strain regained the ability to generate a blue coloration.we analyzed the transcriptional levels of lux I and lux R genes in the three mutations?ssp A,hns,rpo S?.The relative expression of lux I and lux R increased significantly in?hns strain,while decreased significantly in?rpo S and?ssp A strains.Based on these data,we can conclude that SspA and RpoS positively regulate the QS system,while H-NS negatively regulates the QS system.To investigate the relationship between SspA,H-NS and RpoS in regulating the QS system,we found that the relative expression of rpo S in?hns strain increased significantly,while the relative expression of hns in?rpo S strains did not change much.Moreover,the relative expression of rpo S in?ssp A strain decreased significantly.In contrast,the relative expression of hns increased significantly.The relative expression of ssp A in?hns and?rpo S strains didn't have significant changes.In order to further verify the above regulatory relationship,we used the Lac Z reporter system to detect the promoter activities of rpo S and hns respectively,the rpo S promoter activity is significantly increased in the?hns.In addition,the activity of hns promoter is significantly increased in?ssp A.Therefore,SspA negatively regulates hns,H-NS negatively regulates rpo S,while SspA positively regulates rpo S.H-NS and RpoS have no regulatory effect on ssp A.Sequence analysis showed that there was a“lux-box”in the upstream region of the ssp A gene.By analyzing transcription level,we found that the expression of ssp A and rpo S was dramatically reduced in the absence of lux R,while the relative expression levels of hns were significantly up-regulated.In?lux R,the activity of P_{ssp A} also decreased significantly.It is speculated that Lux R positively regulates ssp A at the transcription level by binding the“lux-box”upstream of ssp A gene,thereby negatively regulating hns,and positively regulating rpo S.Based on the data,we take as a hypothesis that SspA regulates the QS system through three ways:?1?SspA regulates lux IR,and Lux R also regulates ssp A through the“lux-box”upstream of ssp A,and there is an interaction between the two;?2?SspA indirectly positively regulates lux IR through H-NS.SspA negatively regulates hns,H-NS negatively regulates lux IR;?3?H-NS negatively regulates rpo S and RpoS positively regulates lux IR,so that ssp A regulates lux IR indirectly.