A Novel Transcription Factor VPA0041 Was Identified to Regulate the Swarming Motility in Vibrio parahaemolyticus
Abstract
:1. Introduction
2. Results
2.1. VPA0041 Regulated the Swarming Motility of V. parahaemolyticus
2.2. The Annotation of VPA0041 Protein in V. parahaemolyticus
2.3. The Global Transcriptional Analysis of VPA0041 in V. parahaemolyticus
2.4. VPA0041 Regulates the Expression of the Lateral Flagellar Genes
2.5. VPA0041 Directly Bound to the Promoters of vpa0264, vpa1548, and vpa1550 to Activate the Lateral Flagellar System
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, and Growth Conditions
4.2. Construction of vpa0041 Mutant and Complemented Strains
4.3. The Motility Analysis
4.4. The Transmission Electron Microscope Analysis
4.5. qRT-PCR Analysis of the Lateral Flagellar Genes
4.6. RNA-Seq Analysis
4.7. Purification of VPA0041 Protein
4.8. Electrophoretic Mobility Shift Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strain or Plasmid | Relevant Characteristics | Reference |
---|---|---|
E. coli | ||
DH5α λpir | Host for π requiring plasmids | [53] |
SM10 λpir | Host for π requiring plasmids, conjugal donor | [54] |
BL21(DE3) | Host strain for protein expression | Novagen |
V. parahaemolyticus | ||
RIMD 2210633 | Clinical isolate. Carbr | [23] |
∆vpa0041 | RIMD 2210633, in-frame deletion in vpa0041, Carbr | This study |
vpa0041+ | ∆vpa0041, pMMB207 expressing the vpa0041-his gene, Carbr, Cmr | This study |
Plasmids | ||
pDM4 | Suicide vector, pir dependent, R6K, SacBR, Cmr | [55] |
pMMB207 | IncQ lacIq Δbla Ptac-lac lacZa, Cmr | [56] |
pET30a | Expressing vector, Kmr | Novagen |
vpa0041::pDM4 | vpa0041 up and down sequences clones into pDM4, Cmr | This study |
vpa0041::pMMB207 | RBS and vpa0041-his sequences clones into pMMB207, Cmr | This study |
vpa0041::pET30a | vpa0041 ORF clones into pET30a, Kmr | This study |
Primer Name | Primer Sequence (5′ to 3′) | Target |
---|---|---|
vpa0041-up-F | GATAACAATTTGTGGAATCCCGGGAAGAAGAAATGGGTCAGAAGCGTT | vpa0041 deletion mutant |
vpa0041-up-R | CGTCCGATGACTTAACATCCATAAACCAACTCCTG | vpa0041 deletion mutant |
vpa0041-down-F | GGATGTTAAGTCATCGGACGCAAACCATTAAC | vpa0041 deletion mutant |
vpa0041-down-R | AGTGTATATCAAGCTTATCGATACCCGCACAAAGACAGTGAAGGCAAT | vpa0041 deletion mutant |
vpa0041-out-F | TTGTTGCGACCGTTTAGCGTATG | vpa0041 deletion mutant |
vpa0041-out-R | CGCACAAAGACAGTGAAGGCAAT | vpa0041 deletion mutant |
vpa0041-in-F | TACGCAAGCAGCGGTCAGTG | vpa0041 deletion mutant |
vpa0041-in-R | GCTCAGAAGCGAAGCGTGTT | vpa0041 deletion mutant |
pDM4-F | GGTGCTCCAGTGGCTTCTGTTTCTA | vpa0041 deletion mutant |
pDM4-R | CAGCAACTTAAATAGCCTCTAAT | vpa0041 deletion mutant |
vpa0041-F | CCGCGAGCTCTAAGGAGGTAGGATAATAATGGATGTTAAGGTCTTTAGAACAT | vpa0041+ |
vpa0041-R | GCGTCGACTTAGTGATGATGATGATGATGATGGTTTGCGTCCGATGATGC | vpa0041+ |
pMMB207-F | CTCCCGTTCTGGATAATGTTTTTTG | vpa0041+ |
pMMB207-R | TCTTCTCTCATCCGCCAAAACAGCC | vpa0041+ |
vpa0041-his-F | GGAATTCCATATGATGGATGTTAAGGTCTTTAGAAC | VPA0041 expression |
vpa0041-his-R | GGAATTCCATATGATGGATGTTAAGGTCTTTAGAAC | VPA0041 expression |
pET30a-F | TAGTTATTGCTCAGCGGTGGC | VPA0041 expression |
pET30a-R | ACGATGCGTCCGGCGTAGAG | VPA0041 expression |
gyrb-RT-F | TTACCGTCATGGTGAGCCTG | qRT-PCR |
gyrb-RT-R | CACGCAGACGTTTTGCTAGG | qRT-PCR |
vpa0264-RT-F | GCAGGTTCAGGCCCAGTATT | qRT-PCR |
vpa0264-RT-R | TCATGTTGAGAAACGTCAGGCT | qRT-PCR |
vpa1537-RT-F | CGCTTGAGAAAACGACAGTGG | qRT-PCR |
vpa1537-RT-R | CCTACTAATGCGGTCTCGGC | qRT-PCR |
vpa1538-RT-F | CACGTACGCACATATCCGGT | qRT-PCR |
vpa1538-RT-R | ACGAACACCTTGCTCAACCT | qRT-PCR |
vpa1539-RT-F | ATTAGTGAGGGTGCGCCTTT | qRT-PCR |
vpa1539-RT-R | GGTGAAGGGAAGGAATGGCA | qRT-PCR |
vpa1540-RT-F | CAACGCCAGTTCGTCTTAACG | qRT-PCR |
vpa1540-RT-R | ACGGCCAGTAAAGAGAGGTTG | qRT-PCR |
vpa1548-RT-F | GCTGGTGGCCTTATCGAAGA | qRT-PCR |
vpa1548-RT-R | TACTGCGAAGTCTGCATCCAT | qRT-PCR |
FAM-F | FAM-TGCCTGCAGGTCGACGAT | EMSA |
gyrB-EMSA-F | TGCCTGCAGGTCGACGATTGCCTGCAGGTCGACGATGCGCGCG | EMSA |
gyrB-EMSA-R | TGCCAGCGCACCGCTGACCGCAG | EMSA |
vpa0264-EMSA-1F | TGCCTGCAGGTCGACGATGTTTGTCCTGTCGAAAGAATTC | EMSA |
vpa0264-EMSA-1R | CATGCATCTTTCCTTACAGTCGGCT | EMSA |
vpa0264-EMSA-2F | TGCCTGCAGGTCGACGATTAGAGTTTTCCCCCTAATTTT | EMSA |
vpa0264-EMSA-3F | TGCCTGCAGGTCGACGATTCCACTCTTGTTTGTAAGTCAT | EMSA |
vpa0264-EMSA-4F | TGCCTGCAGGTCGACGATGTATCTTGTTTGTATCTTGGCG | EMSA |
vpa1548-EMSA-1F | TGCCTGCAGGTCGACGATGCCGATCAAAGCACATCGGAA | EMSA |
vpa1548-EMSA-1R | CATCTTAGTCTCCTTAGTTTATCAC | EMSA |
vpa1548-EMSA-2F | TGCCTGCAGGTCGACGATCTTAGTGGAATGCAAGTCACT | EMSA |
vpa1548-EMSA-3F | TGCCTGCAGGTCGACGATAAATTTTTAATTTTCAAATTA | EMSA |
vpa1548-EMSA-4F | TGCCTGCAGGTCGACGATAGTGACTAGGGAATATCCCAAG | EMSA |
vpa1550-EMSA-1F | TGCCTGCAGGTCGACGATTTAGTCGCCTTGTCTTATAGGGA | EMSA |
vpa1550-EMSA-1R | CACGAGCTTACTCCCTCTCTCATTG | EMSA |
vpa1550-EMSA-2F | TGCCTGCAGGTCGACGATGGAACACATAAGGTGGAAAATAC | EMSA |
vpa1550-EMSA-3F | TGCCTGCAGGTCGACGATCAAGTCAATGTTTTAAAAGAAT | EMSA |
vpa1550-EMSA-4F | TGCCTGCAGGTCGACGATAGACATACTTTCAAGGCATAGAG | EMSA |
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Li, M.; Meng, H.; Li, Y.; Gu, D. A Novel Transcription Factor VPA0041 Was Identified to Regulate the Swarming Motility in Vibrio parahaemolyticus. Pathogens 2022, 11, 453. https://doi.org/10.3390/pathogens11040453
Li M, Meng H, Li Y, Gu D. A Novel Transcription Factor VPA0041 Was Identified to Regulate the Swarming Motility in Vibrio parahaemolyticus. Pathogens. 2022; 11(4):453. https://doi.org/10.3390/pathogens11040453
Chicago/Turabian StyleLi, Mingzhu, Hongmei Meng, Yang Li, and Dan Gu. 2022. "A Novel Transcription Factor VPA0041 Was Identified to Regulate the Swarming Motility in Vibrio parahaemolyticus" Pathogens 11, no. 4: 453. https://doi.org/10.3390/pathogens11040453