Construction and Biological Characteristics of Lon Gene Deletion Strain of Glaesserella parasuis

doi:10.7668/hbnxb.20195705

Abstract

Abstract:

The aim is to explore the biological function of the Lon gene in Glaesserella parasuis and its impact on the pathogenicity of the bacterium.A fusion gene fragment containing the homologous arms of the Lon gene and the kanamycin resistance gene selection marker was amplified using overlapping PCR,and a recombinant suicide plasmid pToPo-LR-Kana containing this gene fragment was constructed.The pToPo-LR-Kana was transformed into the parent strain ZJ1208 using natural transformation.The Lon gene-deleted strain ZJ1208-ΔLon was identified by kanamycin resistance screening,PCR,and sequencing.The differences between the wild-type strain ZJ1208 and the gene-deleted strain ZJ1208-ΔLon were compared through determination of growth rate,observation by transmission electron microscopy,stress resistance test,ultraviolet resistance test,serum resistance test,the biofilm formation assay,determination of capsule polysaccharide content and virulence test.The results showed that the Lon gene deletion strain ZJ1208-ΔLon was successfully obtained.The deletion strain had a significantly longer cell size,but its growth rate and number of outer membrane vesicles were similar to those of the wild-type strain ZJ1208.ZJ1208-ΔLon had significantly decreased tolerance to osmotic stress,oxidative stress,and heat stress,as well as significantly decreased resistance to ultraviolet radiation. Compared with the parent strain ZJ1208,the biofilm production capacity of ZJ1208-ΔLon and ZJ1208 was similar,but the capsular polysaccharide content of ZJ1208-ΔLon increased significantly,and the serum resistance and virulence in mice decreased significantly.The above results indicate that the deletion of Lon gene has an impact on multiple biological characteristics of G.parasuis,providing new information for further analysis of the biological function of Lon gene in G.parasuis.

Key words: Glaeserella parasuis, ΔLon deficient strain, Natural transformation, Pathogenicity, Biological characteristics

CLC Number:

S858.28

HUANG Ling, SUN Hongchao, YE Shiyi, SU Fei, YUAN Xiufang, XU Lihua, YU Bin, ZHANG Hui, LI Junxing. Construction and Biological Characteristics of Lon Gene Deletion Strain of Glaesserella parasuis[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 222-229. doi: 10.7668/hbnxb.20195705.

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Figures/Tables 11

Tab.1 Primers used in this study

引物名称 Name of the primers	引物序列(5'—3') Primer sequence(5'—3')	目的片段/bp Product size
Lon-U-F-BamHⅠ	GAG GGATCCTAAGATAGGTATAGTTACCAAAGTG	93
Lon-U-R	CCGTTGAATATGGCTCATTGGATTGCTCTCCTAAAATAGAATT
Lon-Kana-F	TTAGGAGAGCAATCCAATGAGCCATATTCAACGGGAAACGTCT	846
Lon-Kana-R	CTAATATCAACTATTTAGAAAAACTCATCGAGCATCAAATGAAAC
Lon-D-F	TCGATGAGTTTTTCTAAATAGTTGATATTAGCTAAGGTGAC	858
Lon-D-R-Hind Ⅲ	GGG CTCGAGCGCAACATTCCGCCATTTTGGAAGG

Fig.1 Construction and verification of deletion strain A.PCR product of homologous arms and drug resistance gene; B.Overlap PCR product of fusion gene fragment; C.Double enzyme digestion identification of pToPo-LR-Kana; D.ZJ1208-ΔLon PCR identification.1.Lon-UP (893 bp); 2.Kana (846 bp); 3.Lon-Down(858 bp); 4.Lon-UP+Kana+Lon-Down(2 597 bp); 5.The vector after double enzyme digestion is 3 000 bp, and the target gene fragment is 2 597 bp; 6.Untreated recombinant plasmid 5 597 bp; 7.ZJ1208-ΔLon Kana PCR amplification fragment (846 bp); 8.ZJ1208-ΔLon Lon PCR amplification fragment; M1.1 000 bp Marker; M2, M3.5 000 bp Marker; M4.2 000 bp Marker.

Fig.2 Colony morphology and bacterial Gram staining of deletion strain and wild strain A,B.ZJ1208-ΔLon deficient strain;C,D.ZJ1208 wild-type strain.

Fig.3 Transmission electron microscopic observation of the deletion strain and wild strain A.ZJ1208-ΔLon gene deletion strain;B.ZJ1208 wild-type strain.

Fig.4 The growth rate curves of ZJ1208-ΔLon deficient strain and ZJ1208 wild-type strain

Fig.5 The biofilm formation of ZJ1208-ΔLon deficient strain and ZJ1208 wild-type strain Different lowercase letters indicate significant differences between different treatments at P<0.05 level.The same as Fig.6-8.

Fig.6 Effect of Lon gene on stress resistance of ZJ1208 strain A.Osmotic stress;B.Oxidation stress;C.Heat shock stress.

Fig.7 SDS-PAGE electrophoresis and quantification of capsule polysaccharides A.SDS-PAGE of ZJ1208 wild-type strain and ZJ1208-ΔLon deletion strain;B.Capsular polysaccharide expression between ZJ1208 wild strain and ZJ1208-ΔLon deletion strain.

Fig.8 Detection of survival rate of G.parasuis in porcine serum

Fig.9 UV resistance detection of ZJ1208 and ZJ1208-ΔLon

Fig.10 Visceral lesions and survival curves in mice A.Group of ZJ1208-ΔLon;B.Group of ZJ1208;C.Normal mice viscera;D.Survival curves of three groups of mice.

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