New Cloning Vectors to Facilitate Quick Allelic Exchange in Gram-Negative Bacteria

Mejia-Santana, Adrian; Lloyd, Cameron J.; Klose, Karl E.

doi:10.2144/btn-2020-0135

Cited by 3 publications

(3 citation statements)

References 9 publications

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“…V. cholerae mutants were generated through allelic exchange using the plasmids pKEK229 ( 43 ) or pKEK2200 ( 44 ) via sacB counterselection. A complete list of primers and plasmids used is provided in SI Appendix , Tables S2 and S3 .…”

Section: Methodsmentioning

confidence: 99%

A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization

Lloyd,

Guo,

Kinrade

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Vibrio cholerae, the causative agent of the disease cholera, is responsible for multiple pandemics. V. cholerae binds to and colonizes the gastrointestinal tract within the human host, as well as various surfaces in the marine environment (e.g., zooplankton) during interepidemic periods. A large adhesin, the Flagellar Regulated Hemagglutinin A (FrhA), enhances binding to erythrocytes and epithelial cells and enhances intestinal colonization. We identified a peptide-binding domain (PBD) within FrhA that mediates hemagglutination, binding to epithelial cells, intestinal colonization, and facilitates biofilm formation. Intriguingly, this domain is also found in the ice-binding protein of the Antarctic bacterium Marinomonas primoryensis , where it mediates binding to diatoms. Peptide inhibitors of the M. primoryensis PBD inhibit V. cholerae binding to human cells as well as to diatoms and inhibit biofilm formation. Moreover, the M. primoryensis PBD inserted into FrhA allows V. cholerae to bind human cells and colonize the intestine and also enhances biofilm formation, demonstrating the interchangeability of the PBD from these bacteria. Importantly, peptide inhibitors of PBD reduce V. cholerae intestinal colonization in infant mice. These studies demonstrate how V. cholerae uses a PBD shared with a diatom-binding Antarctic bacterium to facilitate intestinal colonization in humans and biofilm formation in the environment.

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Section: Methodsmentioning

confidence: 99%

A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization

Lloyd,

Guo,

Kinrade

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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“…This gblock was ligated into pKEK229 to make plasmid pKEK2013. The Ddns deletion strain was constructed by PCR amplifying upstream and downstream fragments, using primer pairs Del dns frag1 F FC 1 Del dns frag1 R and Del dns frag2 F 1 Del dns frag2 R FC, and PCR amplification of pKEK2200 (51) with primer pair universal1 1 universal2, followed by IVA assembly to create plasmid pKEK2360. All plasmid constructs were verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…V. cholerae strains with chromosomal manipulations were mostly constructed by sucrose counterselection utilizing pKEK2200, a derivative of pDS132 (52), as described previously (51). tacp-qstR was introduced into the O395 genome by streptomycin counterselection utilizing pKAS32 (31,53).…”

Section: Methodsmentioning

confidence: 99%

Natural Transformation in a Classical-Biotype Vibrio cholerae Strain

Lloyd

Mejia-Santana

Dalia

et al. 2021

Appl Environ Microbiol

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Vibrio cholerae causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor V. cholerae is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene hapR. HapR and the regulator TfoX control expression of a third competence regulator QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a hapR plasmid. This technique was also used to facilitate co-transformation into O395 of unmarked DNA (ΔlacZ, ΔflaA, ΔflgG) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome. Importance Natural transformation (uptake of exogenous DNA) in V. cholerae has contributed to the evolution of these human pathogens. Classical biotype V. cholerae were responsible for the first six cholera pandemics, but were replaced by El Tor biotype V. cholerae in the current pandemic. This study demonstrates that classical V. cholerae is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than El Tor V. cholerae suggest the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.

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New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

Van Allen,

Chandrashekarappa,

Bina

et al. 2025

Journal of Microbiological Methods

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New Cloning Vectors to Facilitate Quick Allelic Exchange in Gram-Negative Bacteria

Cited by 3 publications

References 9 publications

A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization

A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization

Natural Transformation in a Classical-Biotype Vibrio cholerae Strain

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

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