Whole-Genome Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY Enzyme for Degradation of the Diffusible Signal Factor
Abstract
:1. Introduction
2. Results
2.1. Sequencing and Analysis of the A. lactucae QL-1 Genome
2.2. Identification and Cloning of the Gene Responsible for the Degradation of DSF
2.3. Activity Detection of FadY
2.4. Xcc Expressing FadY Loses Virulence in Planta
3. Discussion
4. Materials and Methods
4.1. Strains, Plasmids, and Media
4.2. Whole-Genome Sequencing and Assembly
4.3. Genome Component Prediction and Gene Function
4.4. Genome-Based Taxonomic Classification Analysis
4.5. Identification of the Gene Responsible for the Inactivation of DSF
4.6. Construction of an in-Frame Deletion Mutant and Complementation
4.7. Expression and Purification of the FadY Protein
4.8. Codon Optimization of FadY
4.9. Determination of FadY Activity In Vitro
4.10. Virulence Tests
4.11. Nucleotide Accession Number
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Genome Characteristics | |
---|---|
Total length (bp) | 3,973,648 |
GC content (%) | 40.04 |
Number of protein-coding genes | 3.707 |
Average length of protein-coding genes (bp) | 940 |
% of Genome (protein-coding genes) | 87.71 |
rRNA genes | 18 |
tRNA genes | 73 |
Repeats | 153 |
% of Genome (repeats) | 0.37 |
Time (min) | FadY + FA | Control | ||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 1 | 2 | 3 | |
0 | 0.565 | 0.586 | 0.578 | 0.934 | 0.887 | 0.936 |
3 | 0.452 | 0.426 | 0.415 | 0.912 | 0.905 | 0.900 |
5 | 0.382 | 0.377 | 0.334 | 0.943 | 0.922 | 0.899 |
10 | 0.221 | 0.233 | 0.221 | 0.878 | 0.925 | 0.910 |
20 | 0.128 | 0.162 | 0.115 | 0.904 | 0.921 | 0.890 |
30 | 0.000 | 0.000 | 0.000 | 0.869 | 0.869 | 0.824 |
Strains or Plasmids | Relevant Genotype or Phenotype | Sources |
---|---|---|
QL-1 strains | ||
QL-1 | Laboratory storage | This study |
∆fadY | fadY deletion mutant of strain QL-1 with 1680-nt internal coding region deleted | This study |
Xcc XC1 | Pathogenic bacteria causing black rot | Lab collection |
Escherichia coli strains | ||
DH5α | spuE44 ∆lacU169(φ80lacZ∆M15) hsdR17λpir recA1 endA1 gyrA96 thi-1 relA1 | Lab collection |
BL21 | F-ompThsdS (rB−mB−) dcm+ Tetr gal (DE3) endA | Lab collection |
pRK2013 | Tra+, Mob-, ColE1-replicon, Kanr, Sper | Lab collection |
Plasmids | ||
pBBR1-MCS5 | Broad host-range cloning vector; Gmr | Lab collection |
pK18mobsacB | Broad-host-range gene replacement vector, sacB,Gmr | Lab collection |
pBBR1-MCS5-fadY | pBBR1-MCS5 containing fadY under control of Plac | This study |
pK18-fadY | pK18mobsacB contain flanking of fadY | This study |
pGEX-6p-1 | GST fusion protein expression vector, Ampr | Invitrogen |
pGEX-fadY | pGEX-6p-1 containing fadY | This study |
Primers | Sequence (5′–3′) | Applications |
---|---|---|
De3487upF | GAGCTCGGTACCCGGGGATCCGGAGCGCCTGGCGATCAT | For amplification of the 5′-region of fadY |
De3487upR | CGGAGATAATGGCATTTAAGTTAATAAAAAAGCGCCTTAGGG | |
De3487dnF | CTTAAATGCCATTATCTCCGATTCGT | For amplification of the 3′-region of fadY |
De3487dnR | CGACGGCCAGTGCCAAGCTTAGTTGATACAACTTGAAGCG | |
Ts3487-F | CTGCATAGTGCCATCCATCAC | For identification of ∆fadY |
Ts3487-R | GCCAAGGCAGGAAAAAGC | |
cfadY-F | GTCGACGGTATCGATAAGCTTAAGCTAGCGTCGGGCAACA | For construction of pBBR1-MCS5-fadY |
cfadY-R | CGCTCTAGAACTAGTGGATCCAAATATAGAAACAAAAAAAGCGCCC | |
pfadY-F | CAGTCAGTCACGATGCGGCCGATGGAAAAGATTTGGTTTGCAGA | For construction of pGEX-6P-1-fadY |
pfadY-R | CCCCTGGGATCCCCGGAATTCTTAGGTTGGTTTACGTAAGTCTTTACG |
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Ye, T.; Zhou, T.; Xu, X.; Zhang, W.; Fan, X.; Mishra, S.; Zhang, L.; Zhou, X.; Chen, S. Whole-Genome Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY Enzyme for Degradation of the Diffusible Signal Factor. Int. J. Mol. Sci. 2020, 21, 6729. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186729
Ye T, Zhou T, Xu X, Zhang W, Fan X, Mishra S, Zhang L, Zhou X, Chen S. Whole-Genome Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY Enzyme for Degradation of the Diffusible Signal Factor. International Journal of Molecular Sciences. 2020; 21(18):6729. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186729
Chicago/Turabian StyleYe, Tian, Tian Zhou, Xudan Xu, Wenping Zhang, Xinghui Fan, Sandhya Mishra, Lianhui Zhang, Xiaofan Zhou, and Shaohua Chen. 2020. "Whole-Genome Sequencing Analysis of Quorum Quenching Bacterial Strain Acinetobacter lactucae QL-1 Identifies the FadY Enzyme for Degradation of the Diffusible Signal Factor" International Journal of Molecular Sciences 21, no. 18: 6729. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21186729