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Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

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Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens.

W T Peng¹, E W Nester

¹Department of Microbiology, University of Washington, Seattle, WA 98195-7242, USA.

|June 19, 2001

Summary

Agrobacterium tumefaciens possesses a novel efflux pump system, AmeABC, regulated by AmeR. This system, particularly its outer membrane component AmeC, is crucial for resistance to antimicrobial agents.

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Area of Science:

Microbiology
Molecular Biology
Bacterial Genetics

Background:

Agrobacterium tumefaciens is a plant pathogen known for its genetic versatility.
Understanding bacterial efflux pumps is vital for combating antimicrobial resistance.

Purpose of the Study:

To identify and characterize novel efflux pump systems in Agrobacterium tumefaciens.
To elucidate the genetic organization and regulatory mechanisms of these systems.

Main Methods:

Gene sequencing and identification of open reading frames (ORFs).
Construction and analysis of gene mutations (ameA, ameB, ameC, ameR).
Susceptibility profiling against detergents and antibiotics.
Reverse transcription PCR (RT-PCR) for operon analysis.

Main Results:

Four ORFs (ameR, ameA, ameB, ameC) were identified, showing homology to Resistance-Nodulation-Cell Division (RND) efflux pump components.
AmeA and AmeB resemble membrane fusion proteins and RND transporters, while AmeC is similar to outer membrane factors.
The ameABC genes form an operon regulated by AmeR, a TetR family transcriptional regulator.
Mutations in ameC significantly increased susceptibility to antimicrobials, while ameR mutations enhanced resistance.
A gene for SAICAR synthetase is co-transcribed with ameR.

Conclusions:

Agrobacterium tumefaciens possesses a functional RND-type efflux pump, AmeABC, with AmeC being essential for its activity.
The AmeR protein acts as a negative regulator of the ameABC operon.
The identified efflux system likely plays a role in the bacterium's defense against toxic compounds and contributes to antimicrobial resistance.