Activity detection of ldh gene promoter of Streptococcus mutans
ZHANG Jia-qin1, HUANG Shan-shan1, 2, XU Qiao-li1, RAO Hui-hua1, MA Xiao-bo1, HUANG Chao-yang1, FANG Li-li1, ZHENG Gang-sen1, SONG Xiu-yu3
1.Department of Clinical Laboratory, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China; 2.The First Clinical Medical College of Fujian Medical University, Fuzhou 350108, China; 3.Xiamen Central Blood Service Station, Xiamen 361003, China
Abstract：We cloned the ldh gene promoter of Streptococcus mutans and explored it's activity. The candidate promoter of S. mutans ldh gene was amplified from S. mutans UA159 chromosome DNA by PCR, then was inserted into pIB107 by BamH I/Xho I to construct β-glucuronidase report plasmids pCKS11. The plasmids pCKS11 were verified by PCR, restriction enzyme and sequencing. After being linearized by Sca I, the plasmids pCKS11 were transformed into S. mutans UA159. Then, the strain was screened out by THY agar contain 300 μg/mL kanamycin. After being confirmed by PCR and sequencing, the GusA activity of homologous recombination β-glucuronidase report strain SCKS11 and it parental strains were measured. Results showed that the 269 bp candidate promoter of ldh gene was amplified successfully. PCR, restrictive endonuclease digest and sequencing confirmed the validity of the ldh-gusA report plasmids and strains. The result of GusA assays showed that the activity of the candidate promoter of S. mutans ldh gene were 5.8 folds of that of the negative control without any promoter and 0.9 folds of that of the clpP gene promoter of S. mutans positive control. The success of locating the promoter of S. mutans ldh gene and construction of report strains offered an experimental basis to the further study of the expression and regulation of ldh gene of S. mutans.
[1] Singh K, Senadheera DB, Levesque CM, et al. The copYAZ operon functions in copper efflux, biofilm formation, genetic transformation, and stress tolerance in Streptococcus mutans [J]. J Bacteriol, 2015, 197(15): 2545-2557. [2] Ahn SJ, Kaspar J, Kim JN, et al. Discovery of novel peptides regulating competence development in Streptococcus mutans [J]. J Bacteriol, 2014, 196(21): 3735-3745. [3] Biswas I, Jha JK, Fromm N. Shuttle expression plasmids for genetic studies in Streptococcus mutans [J]. Microbiology, 2008, 154(Pt 8): 2275-2282. [4] Baker JL, Derr AM, Faustoferri RC, et al. Loss of NADH oxidase activity in Streptococcus mutans leads to rex-mediated overcompensation in NAD+ regeneration by lactate dehydrogenase[J]. J Bacteriol, 2015, 197(23): 3645-3657. [5] Merritt J, Kreth J, Qi F, et al. Non-disruptive, real-time analyses of the metabolic status and viability of Streptococcus mutans cells in response to antimicrobial treatments[J]. J Microbiological Methods, 2005, 61(2): 161-170. [6] Osterberg S, Peso-Santos TD, Shingler V. Regulation of alternative sigma factor use[J]. Annual Rev Microbiol, 2011, 65(1): 37-55. [7] Islam MS, Pallen Mark J, Busby Stephen JW. A cryptic promoter in the LEE1 regulatory region of enterohaemorrhagic Escherichia coli : promoter specificity in AT-rich gene regulatory regions[J]. Biochem J, 2011, 436(3): 681-686. [8] Bron PA, Hoffer SM, Van Swam II, et al. Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe[J]. Appl Environmental Microbiol, 2004, 70(1): 310-317. [9] Zhang J, Banerjee A, Biswas I. Transcription of clpP is enhanced by a unique tandem repeat sequence in Streptococcus mutans [J]. J Bacteriol, 2009, 191(3): 1056-1065.
2016, Vol. 32 
