Draft Genome Sequence of Ralstonia sp. MD27, a Poly(3-Hydroxybutyrate)-Degrading Bacterium, Isolated from Compost.

Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb.

GENOME ANNOUNCEMENT

Ralstonia species are a diverse group that includes plant and human pathogens and other soil-dwelling organisms, such as Ralstonia eutropha (now known as Cupriavidus necator) (1), Ralstonia solanacearum (2), and Ralstonia pickettii (3, 4). Ralstonia and related species like Cupriavidus necator synthesize and mobilize poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoate (PHA) biopolymers as part of their natural metabolism (5, 6). PHA has attracted much recent interest as a potential biobased, biodegradable alternative to petroleum-based plastics (7, 8).

Ralstonia sp. strain MD27 was isolated from compost from the greenhouse at Wellesley College (Wellesley, MA, USA) using media containing P(3HB) as the sole carbon source. The bacterium grows using P(3HB) with a doubling time of 3.3 h. Genomic DNA of Ralstonia sp. MD27 was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). Libraries were then generated using the Nextera XT sequencing kit (Illumina, San Diego, CA, USA) and sequenced by an Illumina MiSeq at Tufts University Genomics Core Facility. We obtained 2,298,090 2 × 250-bp reads (185-fold coverage), which were assembled into 72 contigs (from 611 to 600,706 bp) using CLC Genomics Workbench version 7.5.

The draft genome sequence of MD27 comprises 5,917,320 bp (63.6% G+C content) and was annotated using the NCBI PGAP pipeline. We predicted 5,382 protein coding sequences, 47 tRNA sequences, and three copies each of 16S, 23S, and 5S rRNA genes. The 16S rDNA sequence of MD27 was found to be most closely related to that of Ralstonia pickettii strain 12J (99% identity based on nucleotide BLAST).

Two PHA synthase (phaC) genes were predicted (contigs 4 and 6), both of which appear to code for the conserved lipase box necessary for activity (9). We also detected two putative β-ketothiolase (phaA) genes (contigs 6 and 12), and two putative acetoacetyl-CoA reductase (phaB) genes (contigs 4 and 6). Indeed, the phaCAB sequences in contig 6 appear to form a gene cluster similar to a PHA biosynthesis operon found in C. necator H16 (10, 11) and R. pickettii strain 12D. A putative regulatory gene (phaR) has been identified in contig 6. These findings suggest that MD27 can synthesize intracellular polyhydroxyalkanoates.

Four putative PHA depolymerase (phaZ) genes were identified in the MD27 genome: three intracellular depolymerase genes (contigs 2, 6, and 22) and an extracellular P(3HB) depolymerase gene (contig 34) that were predicted, based on a BLAST search with the Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) (12), to be an extracellular short-chain-length PHA depolymerase with a type I catalytic domain, as previously described (13). Experimentally validated PHA depolymerases that also fall into this category include the PhaZ from Ralstonia pickettii (14). Ralstonia sp. MD27 expresses a P(3HB) depolymerase that is capable of breaking down highly crystalline P(3HB) and is unlike the well-studied model organism of PHA homeostasis, C. necator strain H16 (10, 11), implying that Ralstonia sp. MD27 could be utilized in composting items made of P(3HB).

Nucleotide sequence accession numbers.

This whole-genome shotgun sequencing project has been deposited in DDBJ/EMBL/GenBank under accession number LFZM00000000. The version described in this paper is the first version, LFZM01000000.