A Tripartite Efflux System Affects Flagellum Stability in Helicobacter pylori
Abstract
:1. Introduction
2. Results
2.1. Identification of H. pylori Proteins That Are Found Preferentially in Helicobacter Species with Flagellar Sheaths
2.2. HP1486, HP1487 and HP1489 Are Required for Robust Motility of H. pylori
2.3. In-Situ Structures of Flagellar Motors in the Δhp1489 and Δhp1487/hp1486 Mutants Reveal Apparent Flagellum Disassembly Products
2.4. Isolation of Mutants That Suppress the Motility Defect in the Δhp1487/hp1486 Mutant
3. Discussion
3.1. The HP1489-HP1486 Tripartite Efflux System Has an Apparent Role in Stabilizing the H. pylori Flagellar Motor
3.2. Identification of Genes Potentially Involved in Sheath Formation
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
4.2. Strain Construction
4.3. Motility Assay
4.4. Complementation of Δhp1489 Mutation
4.5. Isolation of Variants of ∆hp1487/hp1486 Mutant with Enhanced Motilities
4.6. Genome Sequencing and Analysis
4.7. Transmission Electron Microscopy
4.8. Sample Preparation for Cryo-EM Observation
4.9. Cryo-ET Data Collection and Image Processing
4.10. Sub-Tomogram Analysis with i3 Packages
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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NCBI Reference | Locus Tag | Gene | Known/Proposed Function | Reference |
---|---|---|---|---|
Flagellum function | ||||
WP_000742695.1 | HP0327 | pseH | flagellin glycosylation | [31] |
WP_000868000.1 | HPG27_395 | flgV | required for motility | [32] |
Lipopolysaccharide biosynthesis | ||||
WP_000487418.1 | HP0329 | futA | fucosylation of LPS | [33] |
WP_000433778.1 | HP0580 | kdhA | Kdo-lipid A hydrolase | [34] |
WP_000898474.1 | HP1580 | lpxF | lipid A 4′-phosphatase | [35] |
WP_041201341.1 | HP1581 | wecA | O-antigen assembly | [36] |
Sel1-like proteins | ||||
WP_000597817.1 | HP0160 | hcpD | penicillin-binding protein | [37] |
WP_000901623.1 | HP0211 | hcpA | penicillin-binding protein | [38] |
WP_000111740.1 | HP0235 | hcpE | ||
WP_000917816.1 | HP0628 | hcpF | ||
WP_000892789.1 | HP1098 | hcpC | ||
WP_000540103.1 | HP1117 | |||
WP_000943858.1 | HPG27_1469 | hcpG | ||
Outer membrane proteins | ||||
WP_041201349.1 | HP0209 | hofA | [39] | |
WP_000768629.1 | HP0486 | hofC | [39] | |
WP_012552416.1 | HP0487 | hofD | [39] | |
WP_000911466.1 | HP0782 | hofE | [39] | |
WP_001108270.1 | HP0788 | hofF | [39] | |
WP_041201373.1 | HP0914 | hofG | [39] | |
WP_000797787.1 | HP1083 | hofB | [39] | |
Transport proteins | ||||
WP_000788001.1 | HP0839 | fatty acid transport protein | [39] | |
WP_000816869.1 | HP0970 | cznB | metal efflux pump | [40] |
WP_079990419.1 | HP0971 | cznC | metal efflux pump | [40] |
WP_000780237.1 | HP1028 | lipocalin | [41] | |
WP_001008850.1 | HP1486 | ABC-2 family transporter protein | this study | |
WP_000489110.1 | HP1487 | ABC-2 family transporter protein | this study | |
WP_012552579.1 | HP1488 | membrane fusion protein | this study | |
WP_000754037.1 | HP1489 | outer membrane efflux protein | this study | |
Miscellaneous functions | ||||
WP_000820014.1 | HP0018 | predicted lipoprotein | ||
WP_041201345.1 | HP0097 | predicted lipoprotein | ||
WP_000233964.1 | HP0111 | hrcA | heat-inducible repressor | [42] |
WP_000689112.1 | HP0190 | clsC | cardiolipin synthase | [43] |
WP_001159179.1 | HP0199 | |||
WP_000114771.1 | HP0468 | DUF5644 domain-containing protein | ||
WP_000462324.1 | HP0640 | poly(A) polymerase | ||
WP_000949206.1 | HP0653 | ftnA | bacterial non-heme ferritin | [44] |
WP_000413451.1 | HP0664 | DUF2603 domain-containing protein | ||
WP_001279170.1 | HP0700 | dgkA | diacylglycerol kinase | |
WP_000790557.1 | HP0827 | RNA or ssDNA-binding protein | ||
WP_001268507.1 | HP0838 | putative lipoprotein | ||
WP_012552558.1 | HP1321 | conserved hypothetical ATP-binding protein | ||
WP_000896338.1 | HP1440 | ispDF | isoprenoid biosynthesis | [45] |
Class 1 | Class 2 | Class 3 | Class 4 | |
---|---|---|---|---|
Δhp1489 motor number | 40 | 4 | 18 | 9 |
Δhp1487/hp1486 motor number | 91 | 17 | 15 | 6 |
Isolate | Gene Description | Locus Tag | Sequence a | Impact b | Freq c |
---|---|---|---|---|---|
ESMV1 | arsS | CV725_RS0308 | (G)11→12 coding (1261/1281 nt) | P421fs | 84.9% |
fliL | CV725_04935 | (A)9→8 coding (46/552 nt) | S16fs | 96.6% | |
oipA | CV725_05780 | (AG)9→8 coding (51-52/958 nt) | S18sf | 93.3% | |
glycosyltransferase family 25 protein | CV725_RS05870 | (G)13→14 coding (539/633 nt) | T180fs | 94.3% | |
cag pathogenicity island protein | CV725_RS06350 | 2 bp→AG coding (2657-2658/5466 nt) | N886K | 96.6% | |
ESMV2 | cation:proton antiporter | CV725_RS04480 | GCC→ACC | A249T | 98.9% |
fliL | CV725_04935 | (A)9→8 coding (46/552 nt) | S16fs | 97.3% | |
murJ | CV725_06605 | (A)9→8 coding (1174/1462 nt) | S392fs | 98.6% | |
hypothetical protein | CV725_RS06835 | CTA→CGA | L4R | 100% | |
hypothetical protein | CV725_RS06835 | TTC→TCC | F5S | 100% | |
hypothetical protein | CV725_RS06835 | 2 bp→CT coding (17-18/2007 nt) | I6L | 100% | |
outer membrane beta-barrel protein | CV725_RS07750 | (TC)9→8 pseudogene (14-15/1989 nt) | L5fs | 96.4% | |
glycosyltransferase family 8 protein | CV725_RS03290 | (TC)9→8 pseudogene (95-96/1162 nt) | R28fs | 96.2% | |
ESMV3 | fliL | CV725_04935 | (A)9→8 coding (46/552 nt) | S16fs | 99.7% |
murJ | CV725_06605 | (A)9→8 coding (1174/1462 nt) | S392fs | 99.4% | |
cation:proton antiporter | CV725_RS04480 | GCT→ACT | A201T | 99.1% |
Strain | Relevant Genotype | Source |
B128 | wild-type | [58] |
G27 | wild-type | [59] |
HP116 | H. pylori B128 hp1489:: kanR-PureA-sacB | this study |
HP119 | H. pylori B128 ∆hp1489 | this study |
HP174 | H. pylori B128 hp1487/hp148639:: kanR-PureA-sacB | this study |
HP185 | H. pylori B128 ∆hp1487/hp1486 | this study |
HP121 | H. pylori G27 hp1489:: kanR-PureA-sacB | this study |
HP122 | H. pylori G27 ∆hp1489 | this study |
HP128 | HP122 bearing pHel1489 | this study |
Plasmid | Description | Source |
pGEM-T Easy | TA cloning vector; ampR | Promega |
pJC038 | pGEM-T Easy carrying kanR-PureA-sacB | [43] |
pJC049 | pGEM-T Easy carrying flanking regions of hp1489 | this study |
pJC051 | pJC049 with kanR-PureA-sacB insertion | this study |
pJC076 | pGEM-T Easy carrying flanking regions of hp1487/hp1486 | this study |
pJC080 | pJC076 with kanR-PureA-sacB insertion | this study |
pJC083 | pGEM-T Easy carrying Php1491-hp1489 | this study |
pHel3 | H. pylori shuttle vector; kanR | [60] |
pHel1489 | pHel3 carrying hp1489 | this study |
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Gibson, K.; Chu, J.K.; Zhu, S.; Nguyen, D.; Mrázek, J.; Liu, J.; Hoover, T.R. A Tripartite Efflux System Affects Flagellum Stability in Helicobacter pylori. Int. J. Mol. Sci. 2022, 23, 11609. https://doi.org/10.3390/ijms231911609
Gibson K, Chu JK, Zhu S, Nguyen D, Mrázek J, Liu J, Hoover TR. A Tripartite Efflux System Affects Flagellum Stability in Helicobacter pylori. International Journal of Molecular Sciences. 2022; 23(19):11609. https://doi.org/10.3390/ijms231911609
Chicago/Turabian StyleGibson, Katherine, Joshua K. Chu, Shiwei Zhu, Doreen Nguyen, Jan Mrázek, Jun Liu, and Timothy R. Hoover. 2022. "A Tripartite Efflux System Affects Flagellum Stability in Helicobacter pylori" International Journal of Molecular Sciences 23, no. 19: 11609. https://doi.org/10.3390/ijms231911609