Screening Quorum-sensing-associated sRNA and construction of its overexpression and deletion mutant in Pseudomonas aeruginosa
10.16571/j.cnki.1008-8199.2020.04.003
- VernacularTitle: 铜绿假单胞菌群体感应相关sRNA的筛选及突变株的构建
- Author:
Hong-lin LI
1
;
Yang LU
2
;
Yu-yang LIU
3
;
She-bin ZHANG
1
;
Yi-mei CAI
3
;
Jian-ming ZENG
2
;
Bin HUANG
3
;
Cha CHEN
2
Author Information
1. The Second Clinical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China
2. Department of Laboratory Medicine,the Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China
3. Deptartment of Laboratory Medicine,First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080, Guangdong, China
- Publication Type:Journal Article
- Keywords:
Pseudomonas aeruginosa;
quorum-sensing system;
sRNA;
RNA-seq;
overexpression;
knockout
- From:
Journal of Medical Postgraduates
2020;33(4):348-355
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveQuorum-sensing (QS) and small regulatory RNA (sRNA) play key regulatory roles in many signaling cascades of Pseudomonas aeruginosa. To investigate whether sRNA is involved in P. aeruginosa QS system, screening QS system-related sRNA, and to construct sRNA overexpression and deletion strains of Pseudomonas aeruginosa for further study of sRNA function.MethodsSRNA associated with the QS system was screened by qPCR and RNA-sequencing (RNA-seq). The target gene were amplified by PCR and inserted into the overexpression vector pROp200 or the homologous recombination vector pGSM-MR, respectively. The connection reaction solution of pROp200-sRNA and pGSM-ΔsRNA was transformed into Escherichia coli DH5a and SM10lp, respectively. The recombinant vectors were identified by PCR. The pROp200-sRNA was transformed into PAO1 by heat shock method, and the pGSM-ΔsRNA was transferred from SM10lp to PAO1 by conjugation. SRNA overexpression and deletion strains were identified by PCR, DNA sequencing and qPCR, the determination of the growth curves and the pyocyanin levels of strains.ResultsFive QS -associated sRNA P26, P5316.1, P30, P34 and AmiL were successfully screened by RNA-seq and qPCR. PCR, DNA sequencing and qPCR showed that sRNA of AmiL, P30 and P34 overexpression and knockout were successful. Compared with wild-type strain, sRNA overexpression and knockout had no significant effect on bacterial growth curve. It were notably that overexpression of AmiL and P30 inhibited and increase the production of pyocyanin, respectively (P<0.01), while deficiency of AmiL and P30 in the genome resulted in significantly increase and decrease of pyocyanin, respectively (P<0.01). Overexpression and deletion of P34 had no significant effect on pyocyanin synthesis (P>0.05).ConclusionThe sRNA overexpression and deletion strains have been successfully constructed and can be used to study the regulatory relationship between sRNA and QS systems, and to further functional study.
