A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation
Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2018-01-01
|
| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/4253.pdf |
| _version_ | 1797419611483799552 |
|---|---|
| author | Ke Dai Xintian Wen Yung-Fu Chang Sanjie Cao Qin Zhao Xiaobo Huang Rui Wu Yong Huang Qigui Yan Xinfeng Han Xiaoping Ma Yiping Wen |
| author_facet | Ke Dai Xintian Wen Yung-Fu Chang Sanjie Cao Qin Zhao Xiaobo Huang Rui Wu Yong Huang Qigui Yan Xinfeng Han Xiaoping Ma Yiping Wen |
| author_sort | Ke Dai |
| collection | DOAJ |
| description | Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R) or at 88th (AAA → AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively. |
| first_indexed | 2024-03-09T06:50:47Z |
| format | Article |
| id | doaj.art-04ba360b1ae14d88b7790ca3b1e2771e |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:50:47Z |
| publishDate | 2018-01-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-04ba360b1ae14d88b7790ca3b1e2771e2023-12-03T10:27:20ZengPeerJ Inc.PeerJ2167-83592018-01-016e425310.7717/peerj.4253A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformationKe Dai0Xintian Wen1Yung-Fu Chang2Sanjie Cao3Qin Zhao4Xiaobo Huang5Rui Wu6Yong Huang7Qigui Yan8Xinfeng Han9Xiaoping Ma10Yiping Wen11College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaDepartment of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of AmericaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaCollege of Veterinary Medicine, Sichuan Agricultural University, Chengdu, ChinaHaemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R) or at 88th (AAA → AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively.https://peerj.com/articles/4253.pdfrpsLPoint mutationEMSHaemophilus parasuisStreptomycin resistanceNatural transformation |
| spellingShingle | Ke Dai Xintian Wen Yung-Fu Chang Sanjie Cao Qin Zhao Xiaobo Huang Rui Wu Yong Huang Qigui Yan Xinfeng Han Xiaoping Ma Yiping Wen A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation PeerJ rpsL Point mutation EMS Haemophilus parasuis Streptomycin resistance Natural transformation |
| title | A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation |
| title_full | A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation |
| title_fullStr | A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation |
| title_full_unstemmed | A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation |
| title_short | A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation |
| title_sort | streptomycin resistance marker in h parasuis based on site directed mutations in rpsl gene to perform unmarked in frame mutations and to verify natural transformation |
| topic | rpsL Point mutation EMS Haemophilus parasuis Streptomycin resistance Natural transformation |
| url | https://peerj.com/articles/4253.pdf |
| work_keys_str_mv | AT kedai astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xintianwen astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yungfuchang astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT sanjiecao astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT qinzhao astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xiaobohuang astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT ruiwu astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yonghuang astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT qiguiyan astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xinfenghan astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xiaopingma astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yipingwen astreptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT kedai streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xintianwen streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yungfuchang streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT sanjiecao streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT qinzhao streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xiaobohuang streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT ruiwu streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yonghuang streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT qiguiyan streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xinfenghan streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT xiaopingma streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation AT yipingwen streptomycinresistancemarkerinhparasuisbasedonsitedirectedmutationsinrpslgenetoperformunmarkedinframemutationsandtoverifynaturaltransformation |