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Mutagenesis, cloning and complementation analysis of C4 -dicarboxylate transport genes from Rhodobacter capsulatus
M J Hamblin1, J G Shaw1, J P Curson1
1Robert Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN. UK.
Molecular Microbiology
|August 5, 2017
Summary
Researchers identified essential genes for C4-dicarboxylate transport in Rhodobacter capsulatus. Mutants lacking this transport system showed impaired aerobic growth but retained photoheterotrophic capabilities, highlighting the dicarboxylate transporter
Area of Science:
- Microbiology
- Bacterial Physiology
- Molecular Genetics
Background:
- Rhodobacter capsulatus is a photosynthetic bacterium with diverse metabolic capabilities.
- Efficient nutrient uptake is crucial for bacterial growth and survival.
- Understanding carbon metabolism pathways is key to microbial biotechnology.
Purpose of the Study:
- To identify genes responsible for C4-dicarboxylate transport in Rhodobacter capsulatus.
- To investigate the role of C4-dicarboxylate transport in aerobic and photoheterotrophic growth.
- To characterize the genetic locus involved in dicarboxylate transport.
Main Methods:
- Transposon mutagenesis to generate mutants.
- Growth assays under various aerobic and photoheterotrophic conditions.
- Complementation analysis using a cosmid gene bank.
- Subcloning to define the essential genetic region.
Main Results:
- Five mutants unable to grow aerobically on malate, succinate, or fumarate were isolated.
- All mutants exhibited deficiencies in C4-dicarboxylate transport.
- Mutants retained photoheterotrophic growth on malate and succinate, but not fumarate.
- Complementation identified a mutated locus (dct) containing at least three linked genes.
Conclusions:
- C4-dicarboxylate transport is essential for aerobic growth on these carbon sources in Rhodobacter capsulatus.
- The dct locus, spanning 8.3kb, harbors genes critical for aerobic dicarboxylate transport.
- Rhodobacter capsulatus possesses alternative pathways for photoheterotrophic growth independent of aerobic dicarboxylate transport.
