Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel

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.

Fig 1

Comparison of the hydrolytic degradation pathways of melamine and atrazine. The genes encoding microbial enzymes that catalyze each step are indicated. All of the enzymes catalyzing the first three steps of both pathways are members of the amidohydrolase superfamily (11, 26, 31, 32). The first step after the pathways converge is catalyzed by cyanuric acid hydrolases (13), which are in an uncharacterized family that includes members with only one other known functionality (barbiturases) (34). The known biuret deaminating enzymes belong to two different superfamilies: AtzE and TrzF are in the amidase signature superfamily, and pRL100352 encodes a cysteine hydrolase superfamily biuret hydrolase in Rhizobium leguminosarum bv. viciae 3841 (3). The known microbial allophanate hydrolases are also members of the amidase signature superfamily. Superscript numbers: 1, GDA is an abbreviation of guanine deaminase; 2, ammeline deamination by AtzB was reported in one study (26) but was not observed in a later study (28); 3, Moth-2120 encodes a cyanuric acid hydrolase in Moorella thermoacetica ATCC 39073 (19).

Fig 2

Growth of Rhodococcus sp. strain Mel on melamine or NH₄Cl as the sole nitrogen source. ▲, no nitrogen control; □, 6 mM NH₄Cl; ●, 1.0 mM melamine. Values are means of data for triplicate samples, and bars are standard deviations. OD₆₀₀, optical density at 600 nm.

Fig 3

Degradation of melamine by Rhodococcus sp. strain Mel. (A) Growth and melamine degradation curves. ▲, growth, OD₆₀₀; ○, melamine concentration in uninoculated control; □, melamine concentration during growth of Rhodococcus sp. strain Mel. (B) HPLC analysis of melamine and its degradation intermediates. □, melamine concentration during growth of Rhodococcus sp. strain Mel; ■, concentration of ammeline; △, concentration of ammelide; ♦, concentration of cyanuric acid. Values are means of data for triplicate samples.

Fig 4

Genomic organization of genes involved in melamine degradation in Rhodococcus sp. strain Mel. (A) Plasmid-localized 22.3-kb contig 305 containing a functional trzA and a potential ammelide deaminase (ammelide aminohydrolase [AAH]). The putative cyanuric acid hydrolase (CAH) and isochorismatase [iso] (potential biuret hydrolase [BH]) did not encode functional melamine pathway enzymes. The putative cytosine deaminase (CDase) was not active on ammeline or ammelide, and the putative allophanate hydrolase (AH) was not functionally characterized. (B) Plasmid-localized 17.9-kb gene region (contig 37) from Rhodococcus sp. strain Mel containing a functional cyanuric acid hydrolase (CAH), biuret hydrolase ([iso]), and mercury resistance genes. The allophanate hydrolase (AH) homolog was not functionally characterized.

Fig 5

Pulsed-field gel electrophoresis of plasmid DNA from Rhodococcus sp. strain Mel. (A) Gel showing the presence of two large indigenous plasmids of ∼265 kb (pMel2) and ∼630 kb (pMel1). (B) Gel showing plasmids in wild-type Rhodococcus sp. strain Mel and acridine orange-cured mutants that lost the ability to grow on melamine, ammelide, cyanuric acid, or biuret as N sources.