Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in <i>Mycobacterium smegmatis</i>

doi:10.13523/j.cb.2103030

China Biotechnology

2021, Vol. 41

Issue (6): 13-22 DOI: 10.13523/j.cb.2103030

Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in Mycobacterium smegmatis

BAI Jia-cheng¹,CHI Ming-zhe¹,HU Ya-wen¹,HAO Meng²,ZHANG Xue-lian^1,^**(

)

1 State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China
2 The Fourth Hospital of Ningxia Hui Autonomous Region, Yinchuan 750021, China

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Abstract

Protein homeostasis through protein quality control is critical for bacterial growth and adaptation to host immune stress. The Clp protease plays an important role in protein degradation and homeostasis in Mycobacterium tuberculosis. There are two proteins in the Clp protease that are responsible for recognizing unfolding substrate proteins:ClpC and ClpX. Then unfoled substract is transferred into the chamber of ClpP, where proteolysis is carried out by protease. In order to explore the respective functional characteristics of ClpC and ClpX in mycobacterium,Mycobacterium smegmatis was selected as the experimental strain, and knockdown strains of ClpC and ClpX were constructed successfully by the CRISPRi method. The results showed that the ClpC and ClpX knockdown strains showed significant growth phenotypes differences compared with wild Mycobacterium smegmatis. Low expression of both ClpC and ClpX severely affected the growth of Mycobacterium smegmatis. ClpC low expression resulted in the loss of biofilm formation ability of the strain, while that of ClpX caused incomplete cell wall and bacterial filamentation indicating that ClpC and ClpX may have different physiological functions in mycobacterium.

Key words： Mycobacterium smegmatis ClpC ClpX

Received: 15 March 2021 Published: 06 July 2021

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Corresponding Authors: Xue-lian ZHANG E-mail: xuelianzhang@fudan.edu.cn

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BAI Jia-cheng,CHI Ming-zhe,HU Ya-wen,HAO Meng,ZHANG Xue-lian. Construction and Biological Characteristics of ClpC and ClpX Knock-down Strains in Mycobacterium smegmatis. China Biotechnology, 2021, 41(6): 13-22.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103030 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I6/13

Table 1 Oligonucleotides used in this study

Table 2 Proteases encoding genes in different bacterial species

Fig.1 Construction and DNA sequencing of recombinant pLJR962 plasmids (a) M:10 000 bp marker; Lane 1:pLJR92 plasmid; Lane2:Digestion product of pLJR962 plasmid by BsmB I (b) Result of DNA sequencing of the recombinant pLJR962-clpC plasmid (c) Result of DNA sequencing of the recombinant pLJR962-clpX plasmid

Fig.2 Verification of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains at the transcriptional level (a) Relative clpC expression of Msm-ΔclpC(KD) strain was decreased compared with Msm∷PLJR962 strain (b) RelativeclpX expression ofMsm-ΔclpX(KD) strain was decreased compared with Msm∷PLJR962 strain All strains were cultured in 7H9-OADC medium with 100 ng/mL ATc. The data presented are expressed as the mean (±s.e.m). Statistical significance was determined usingt-test, *** Represented P<0.001

Fig.3 Growth of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains on solid plate (a) 7H10-OADC plate without ATc (b) 7H10-OADC plate with 100 ng/mL ATc. The first line named 0.1 represents the cell density of OD ₆₀₀=0.1, approximately 10⁷ cells/mL. 2.5 μL bacteria at different density were injected on every grid

Fig.4 Growth curves of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains (a) Growth curve of Msm-ΔclpC(KD) in 7H9-OADC medium with or without 100 ng/mL ATc (b) Growth curve ofMsm-ΔclpX(KD) in 7H9-OADC medium with or without 100 ng/mL ATc

Fig.5 Colony morphology of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains All strains were plated on 7H10-OADC medium with 100 ng/mL ATc

Fig. 6 Biofilm formation of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains

Fig.7 Cell morphology of Msm-ΔclpC(KD) and Msm-ΔclpX(KD) strains


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