Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175

Objective To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function. Method Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinom...

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Autores principales:	LAI Yangfan, WANG Peng, QIAO Li, LIU Zhongjing, YE Zhaoyang, LIANG Yan
Formato:	article
Lenguaje:	ZH
Publicado:	Editorial Department of Journal of Prevention and Treatment for Stomatological Diseases 2021
Materias:	streptococcus mutans ， hit gene， pfw5 vector， natural genetic transformation， homologous recombination， hit-deficient mutant strain， cell growth， cell-cycle regulation， Medicine R
Acceso en línea:	https://doaj.org/article/3a63e10fdf6e403aa69bbd25c6601634
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spelling	oai:doaj.org-article:3a63e10fdf6e403aa69bbd25c66016342021-11-16T03:00:18ZConstruction of the hit-deficient mutant strain of Streptococcus mutans ATCC2517510.12016/j.issn.2096-1456.2021.12.0022096-1456https://doaj.org/article/3a63e10fdf6e403aa69bbd25c66016342021-12-01T00:00:00Zhttp://www.kqjbfz.com/CN/10.12016/j.issn.2096-1456.2021.12.002https://doaj.org/toc/2096-1456Objective To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function. Method Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated. Results The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001). Conclusion The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.LAI Yangfan WANG PengQIAO Li LIU Zhongjing YE ZhaoyangLIANG YanEditorial Department of Journal of Prevention and Treatment for Stomatological Diseasesarticlestreptococcus mutans ，hit gene，pfw5 vector，natural genetic transformation，homologous recombination，hit-deficient mutant strain，cell growth，cell-cycle regulation，MedicineRZH口腔疾病防治, Vol 29, Iss 12, Pp 801-808 (2021)
institution	DOAJ
collection	DOAJ
language	ZH
topic	streptococcus mutans ， hit gene， pfw5 vector， natural genetic transformation， homologous recombination， hit-deficient mutant strain， cell growth， cell-cycle regulation， Medicine R
spellingShingle	streptococcus mutans ， hit gene， pfw5 vector， natural genetic transformation， homologous recombination， hit-deficient mutant strain， cell growth， cell-cycle regulation， Medicine R LAI Yangfan WANG Peng QIAO Li LIU Zhongjing YE Zhaoyang LIANG Yan Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
description	Objective To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function. Method Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated. Results The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001). Conclusion The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.
format	article
author	LAI Yangfan WANG Peng QIAO Li LIU Zhongjing YE Zhaoyang LIANG Yan
author_facet	LAI Yangfan WANG Peng QIAO Li LIU Zhongjing YE Zhaoyang LIANG Yan
author_sort	LAI Yangfan
title	Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
title_short	Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
title_full	Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
title_fullStr	Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
title_full_unstemmed	Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175
title_sort	construction of the hit-deficient mutant strain of streptococcus mutans atcc25175
publisher	Editorial Department of Journal of Prevention and Treatment for Stomatological Diseases
publishDate	2021
url	https://doaj.org/article/3a63e10fdf6e403aa69bbd25c6601634
work_keys_str_mv	AT laiyangfan constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175 AT wangpeng constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175 AT qiaoli constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175 AT liuzhongjing constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175 AT yezhaoyang constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175 AT liangyan constructionofthehitdeficientmutantstrainofstreptococcusmutansatcc25175
_version_	1718426778131759104

Construction of the hit-deficient mutant strain of Streptococcus mutans ATCC25175

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