Journal of Prevention and Treatment for Stomatological Diseases ›› 2021, Vol. 29 ›› Issue (12): 801-808.doi: 10.12016/j.issn.2096-1456.2021.12.002

• Basic Study • Previous Articles     Next Articles

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

LAI Yangfan1(),WANG Peng1,QIAO Li2,LIU Zhongjing2,YE Zhaoyang2(),LIANG Yan1()   

  1. 1. Department of Operative Dentistry and Endodontics, School of Stomatology, Guizhou Medical University, Guiyang 550004, China
    2. Clinical Research Center (Affiliated Hospital), Guizhou Medical University, Guiyang 550004, China
  • Received:2021-02-02 Revised:2021-04-23 Online:2021-12-20 Published:2021-08-17
  • Contact: Zhaoyang YE,Yan LIANG E-mail:1392559217@qq.com;yezy@gmc.edu.cn;519573688@qq.com
  • Supported by:
    grants from Guizhou Science and Technology Fund(ZK[2021]438);Guizhou Health Science and Technology Fund(gzwjkj2019-1-172)

Abstract:

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.

Key words: Streptococcus mutans, hit gene, pFW5 vector, natural genetic transformation, homologous recombination, hit-deficient mutant strain, cell growth, cell-cycle regulation

CLC Number: 

  • R78

Table 1

Primers and sequences used in the experiment"

Primers Sequence (5′-3′)
hit(Up) F:CCGCTCGAGTCAACCCTCTTTAGTCAAAGTCATAT(XhoI)
R:CCCAAGCTTGTCCTCATAAACTTTTGATAGAGGGAATGT(HindIII)
hit(Down) F:GGAATTCCATATGCCTGACTTTACCCGTCTTGGACAAT(NdeI)
R:GGACTAGTAAATAAAAAATAAGGTGGAAGGGTAT(SpeI)
pFW5(PCR) F:AGGATGAGGAGGCAGATTGCCTTGAATAT
R1:ATTCCTCTGACGAATCCATAATGGCTCTT
F1:ATATACGGAAATTATGACTTAGAGGAATT
R:CGCAGCGAGAAAAAAGGCCCACTTTTGT
hit(PCR) F:CATGACGTTTCTTTCTAACTTTGATAT
R:GTTTGTAATACATCTGATAAAGACCGGCT

Figure 1

Electrophoresis of PCR products of the upstream and downstream sequences of the hit gene and the circle map of the pFW5 vector a: DNA electrophoresis of PCR products of the upstream and downstream DNA fragments of the hit gene; M: marker; Up: the upstream DNA fragment of approximately 850 bp; Down: the downstream DNA fragment of approximately 500 bp; b: the vector pFW5 carries two multiple cloning sites, MCS-I and MCS-II, which are separated by the aad9 spectinomycin-resistance gene sequence"

Figure 2

Electrophoresis of PCR- and double digested-products of the recombinant plasmid pFW5 a: DNA electrophoresis of the PCR products of the MCS-Ⅰ- and MCS-Ⅱ- region DNA fragments of the recombinant plasmids pFW5 (20# and 141#); b: DNA electrophoresis of the products (20_x/s and 141_x/s) of the recombinant plasmids pFW5 (20# and 141#) digested by XhoI and SpeI. The pFW5 vector and pFW5_x/s served as the control. M: Marker"

Figure 3

DNA electrophoresis of PCR products of the hit gene DNA fragments from the Streptococcus mutans genome of plasmid (20#) genetic transformation Lanes 20-3, 20-4 to 20-24 correspond to 19 random positive colony forming units on an aad9 spectinomycin-resistant BHI agar plate, respectively"

Figure 4

Electrophoresis and Sanger sequencing of PCR products of the hit gene fragment from the hit-deficient mutant strains and their parental S. mutans ATCC25175 a: DNA electrophoresis of PCR products of the hit gene DNA fragments of the 20-3# and 20-5# strains; b: Sanger forward sequencing chromatograms of the PCR products of 20-3# strain (the top panel) and its parent strain ATCC25175 (the down panel); c: DNA electrophoresis of the PCR products of the hit gene DNA fragments of Streptococcus mutans ATCC25175; d: Sanger reversed sequencing chromatograms of the PCR products of 20-3# strain (the top panel) and its parent strain ATCC25175 (the down panel) "

Figure 5

Electrophoresis and Sanger sequencing chromatograms of the PCR products of the hit gene DNA fragments from the hit-deficient mutant strains a: DNA electrophoresis of PCR products of the hit gene DNA fragments of the 1-1-1-15 and 6-1 strains. b&c: sanger forward sequencing (b) and reversed sequencing chromatograms (c) of the PCR products of 6-1 strains"

Figure 6

Growth curve and growth rate of the hit-deficient mutant strains versus Streptococcus mutans a: the hit-deficient mutant strains (Smu.Δhit) and their parent strains ATCC25175 have similar growth curves; b: the growth rate of the hit-deficient mutant strains (Smu.Δhit) is much faster than that of their parent strains ATCC25175 at 48-hour intervals"

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