Related Experiment Video
Updated: May 26, 2026
Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel
Anthony G Dodge1, Lawrence P Wackett, Michael J Sadowsky
1University of Minnesota, St. Paul, Minnesota, USA.
Abstract:
Rhodococcus sp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase gene trzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ∼265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. The Rhodococcus sp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.
Insights
Rhodococcus sp. strain Mel utilizes melamine as its sole nitrogen source, with key metabolic genes distributed across its genome and a large plasmid. This suggests recent acquisition of melamine degradation capabilities.
Area of Science:
- Microbiology
- Environmental Science
- Biochemistry
Background:
- Melamine is a nitrogen-rich compound with limited natural degradation pathways.
- Microbial degradation of melamine is crucial for nitrogen cycling and remediation.
- Understanding the genetic basis of melamine metabolism in bacteria is essential.
Purpose of the Study:
- To elucidate the genetic and genomic basis of melamine degradation by Rhodococcus sp. strain Mel.
- To identify genes and genetic elements involved in the microbial metabolism of melamine.
- To investigate the role of plasmids in melamine degradation pathways.
Main Methods:
- Isolation and cultivation of Rhodococcus sp. strain Mel using melamine as the sole nitrogen source.
- Whole-genome sequencing using Roche 454 pyrosequencing.
- Gene identification via homology searches (trzA), mutagenesis, and physical mapping (contigs).
- Plasmid analysis through curing and gene transfer experiments.
Main Results:
- Rhodococcus sp. strain Mel efficiently assimilates all six nitrogen atoms from melamine.
- Genes for melamine deamination (trzA homolog) and subsequent hydrolysis (cyanuric acid hydrolase, biuret hydrolase) were identified in distinct genomic regions.
- A self-transmissible linear plasmid (pMel2, ~265 kb) carries four of six essential melamine degradation genes, but is not required for all steps.
- Plasmid-borne genes involved in melamine metabolism appear to be recently acquired.
Conclusions:
- The complete degradation of melamine by Rhodococcus sp. strain Mel involves genes located in at least three separate genomic regions.
- A significant portion of the melamine metabolic pathway is encoded on a mobile genetic element (pMel2), indicating horizontal gene transfer.
- The genetic architecture suggests a dynamic evolutionary history for melamine degradation in this bacterial strain.
