基于结核分枝杆菌牛变种C68001随机突变体库筛选和鉴定成膜相关基因

doi:10.11843/j.issn.0366-6964.2025.08.037

Abstract

Abstract:

This study aimed to utilize a mutant library of Mycobacterium tuberculosis variant (M. tuberculosis var.) bovis C68001 for high-throughput screening to identify key genes affecting biofilm formation. The mutant library of M. tuberculosis var. bovis C68001 was cultured in Sauton′s medium to select mutants with significant differences in biofilm phenotype. The location of mutated genes was determined by resistance marker rescue and sequencing, and the impact of these mutated genes on the antioxidant capacity and SDS resistance of the bacteria was assessed. Furthermore, a phage system was employed to construct strains with target gene deletion, complementation, and overexpression. The effects of the target genes on bacterial mycelium were investigated using scanning electron microscopy, and their influence on bacterial stress resistance and intracellular survival was evaluated by measuring the bacteria′s acid resistance, antioxidant capacity, SDS resistance, and survival within the J774A.1 mouse macrophage cell line. The study successfully screened eight biofilm-deficient strains from the mutant library, with four identified as single-insertion mutants. The mutated genes were Rv1096, Rv3425, Rv3136, and Rv3671c. Knockout, complementation, and overexpression strains of the Rv3671c gene were constructed and verified by PCR and Western blot. These strains exhibited enhanced abilities in biofilm formation, SDS resistance, and intracellular survival. The Rv3671c gene plays a crucial role in the biofilm formation and stress resistance of M. tuberculosis var. bovis C68001. The constructed mutant library of M. tuberculosis var. bovis C68001 provides an efficient tool for target gene screening and identification, offering a reliable approach for exploring the genetic functions of M. tuberculosis var. bovis C68001.

Key words: Mycobacterium tuberculosis variant bovis, Himar 1 transposon, mutant library, biofilm, Rv3671c

CLC Number:

S855.2

SHI Wenjian, XU Lei, ZHANG Ze, YANG Rui, XIN Lingxiang, WANG Nan, CHEN Xiang, XIN Ting. Screening and Identification of Biofilm-related Genes Based on the Random Mutant Library of Mycobacterium tuberculosis Variant bovis C68001[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3992-4006.

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引物名称 Primer name	引物序列(5′→ 3′) Sequence	用途 Function
KanF	GATGGATTGCACGCAGGTTC	转座子插入鉴定
KanR	ATATCACGGGTAGCCAACGC
H1F	CGGGGACTTATCAGCCAACCTG	转座子插入区域侧翼扩增
H1R	TAGAGACCGGGGACTTATCAGCCAA
LFP	TTTTTTTT$\underline{GGCCTAAATGGCC}$CCTGCAAGCGGGAGCGACC	扩增Rv3671c基因左臂
LRP	TTTTTTTT$\underline{GGCCTTTCTGGCC}$CAGCATTGAGCCCAGCGCA
RFP	TTTTTTTT$\underline{CCATAGATTGG}$CAGGTGCTCGGTGTGGTGTTC	扩增Rv3671c基因右臂
RRP	TTTTTTTT$\underline{CCATCTTTTGG}$CGAGGCCGCTGGTCATACTG

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Screening and Identification of Biofilm-related Genes Based on the Random Mutant Library of Mycobacterium tuberculosis Variant bovis C68001

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