Microlunatus phosphovorus NM-1T (= NBRC 101784T)
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Photo by Advanced Industrial Science and Technology (AIST)
Aquatic eutrophication and bloom of toxic algae triggered by release of phosphorus in wastewater have been recognized as environmental problems. The enhanced biological phosphate removal (EBPR) has widely utilized for removing phosphorus from wastewater and preventing phosphorus from diffusing into environment. Polyphosphate accumulating organisms (PAOs) are frequently found in activated sludge in the EBPR process, and believed to play a pivotal role in phosphorus removal. PAOs are microorganisms which take up phosphate into the cells and accumulate it as the form of polyphosphate (polyP) under aerobic conditions. In additions to polyP accumulation, PAOs are supposed to synthesize polyhydroxyalkanoates (PHAs), known as a source of bioplastics. Therefore, PAOs have received industrial attention for their possible use in phosphorus recycling and supply of biodegrative plastics.
Microlunatus phosphovorus NM-1T is an actinobacterium isolated from EBPR activated sludge, as one of rare examples isolated successfully from such an environment. M. phosphovorus NM-1T represents competent features as PAOs; it can accumulate polyP more than 10% of cell mass as phosphorus on a dry weight basis. In addition, a recent study using PHA staining and gas chromatography suggested the presence of PHAs in cells of M. phosphovorus.
The genome of M. phosphovorus NM-1T consisted of a single circular chromosome (5,683,123 bp; G+C content of 67.3%) containing 5,359 predicted protein-coding genes. The M. phosphovorus genome possessed a larger number of genes related to polyP accumulation than other related microorganisms. On the other hand, the homologous genes which are involved in PHA synthesis in proteobacterial PAOs were not observed in M. phosphovorus, suggesting that the organism possibly contains an unknown PHA biosynthetic pathway. Minute analysis of this genome will give useful information and a new insight to investigation about biological mechanisms of polyP accumulation and production of bioplastics.
Project history
2011-06-01 ..... 1
Release of the Microlunatus phosphovorus NM-1T genomic data | We published the genomic data of Microlunatus phosphovorus NM-1T (= NBRC 101784). |
Summary of the genomic data
| Genomic size | 5,683,123 bp | ||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| G+C content | 67.27 % | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Number of ORFs assigned | 5,359 | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Percentage of the coding regions | 90.59 % | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Percentage of the intronic regions | 0.00 % | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Number of rRNA genes |
3
|
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| Number of tRNA genes |
46
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| Number of other features (misc_RNA,misc_feature,repeat) |
0 |
General Procedure
The nucleotide sequence of the M. phosphovorus NM-1T (= NBRC 101784T) genome was determined by the whole genome shotgun sequencing method as in the case of other organisms analyzed at NITE-DOB.
General Procedure
- DNA shotgun libraries
DNA shotgun libraries with inserts of 1.5, 6.0 kb in pUC118 vector (TaKaRa) was constructed.
- Fosmid library
A Fosmid library with inserts of 35 kb in the pCC1FOS fosmid vector was constructed using the CopyControl Fosmid Library Production Kit (Epicentre).
- Nucleotide sequencing
Plasmid and fosmid clones were end-sequenced using dye-terminator chemistry on an ABI 3730xl DNA Analyzer (ABI). Sequence reads were trimmed at a threshold quality value of 20 by Phred and assembled by PHRAP/CONSED software (http://www.phrap.org).
- Gap closing
To close gaps , it selected shotgun or fosmid clones bridging two neighboring contigs and determined their internal sequences by the primer-walking method using custom oligonucleotide primers or the transposon-mediated random insertion method with a Template Generation System II Kit(Finnzyme).
- Validation of the assembled sequence data
The assembled sequence was validated by gel electrophoresis, restriction enzyme digestion of fosmid clone. Finally, each base of M. phosphovorus NM-1 genome was ensured to be sequenced from multiple clones with Phrap quality score ≧ 40.
Gene identification and annotation
- Putative non-translated genes were identified using the Rfam, tRNAscan-SE and ARAGORN programs.
- For the prediction of protein-coding genes, GLIMMER3 program was used. The initial set of ORFs was manually selected from the prediction result in combination with BLASTP.
- Similarity search results against Uniprot, Interpro and HAMAP database were used for functional prediction. The KEGG database was used for pathway reconstruction.
