DNA RESEARCH 19, 383–394, (2012) doi:10.1093/dnares/dss020 Advance Access publication on 23 August 2012 Deciphering the Genome of Polyphosphate Accumulating Actinobacterium Microlunatus phosphovorus AKATSUKI Kawakoshi1,HIDEKAZU Nakazawa1,JUNJI Fukada1,MACHI Sasagawa1,YOKO Katano1, SANAE Nakamura1,AKIRA Hosoyama1,HIROKI Sasaki1,NATSUKO Ichikawa1,SATOSHI Hanada2, YOICHI Kamagata2,KAZUNORI Nakamura2,SHUJI Yamazaki1, and NOBUYUKI Fujita1,* Biological Resource Center, National Institute of Technology and Evaluation, 2-10-49 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan1 and Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan2 *To whom correspondence should be addressed. Tel. þ81 3-6686-2754. Fax. þ81 3-3481-8424. E-mail: [email protected] Edited by Katsumi Isono (Received 25 April 2012; accepted 24 July 2012) Abstract Polyphosphate accumulating organisms (PAOs) belong mostly to Proteobacteria and Actinobacteria and are quite divergent. Under aerobic conditions, they accumulate intracellular polyphosphate (polyP), while they typically synthesize polyhydroxyalkanoates (PHAs) under anaerobic conditions. Many ecological, physiological, and genomic analyses have been performed with proteobacterial PAOs, but few with actino- bacterial PAOs. In this study, the whole genome sequence of an actinobacterial PAO, Microlunatus phos- phovorus NM-1T (NBRC 101784T), was determined. The number of genes for polyP metabolism was greater in M. phosphovorus than in other actinobacteria; it possesses genes for four polyP kinases ( ppks), two polyP-dependent glucokinases ( ppgks), and three phosphate transporters ( pits). In contrast, it harbours only a single ppx gene for exopolyphosphatase, although two copies of ppx are generally present in other actinobacteria. Furthermore, M. phosphovorus lacks the phaABC genes for PHA synthesis and the actP gene encoding an acetate/H1 symporter, both of which play crucial roles in anaerobic PHA accumulation in proteobacterial PAOs. Thus, while the general features of M. phosphovorus regarding aerobic polyP accumulation are similar to those of proteobacterial PAOs, its anaerobic polyP use and PHA synthesis appear to be different. Key words: Microlunatus phosphovorus; whole genome sequence; polyphosphate accumulating organism; polyphosphate; polyhydroxyalkanoate 1. Introduction biological phosphate removal (EBPR) process, where they are believed to play a pivotal role in phosphorus Economically exploitable phosphate rock, the major removal from the wastewater.3 The EBPR process is source of industrial phosphorus, is estimated to be also attracting interest for its potential use in phos- depleted in 50–100 years.1,2 Nevertheless, wasted phorus recycling.4,5 In the EBPR process, PAOs take phosphorus is rarely reused and, to make matters up phosphate into the cells and accumulate it as worse, can induce eutrophication in the surrounding polyphosphate (polyP) under aerobic conditions. In water. Polyphosphate accumulating organisms (PAOs) addition, under subsequent anaerobic conditions, are expected to help solve these problems. PAOs are PAOs in such sludges are thought to accumulate frequently found in activated sludges in the enhanced polyhydroxyalkanoates (PHAs), at the expense of # The Author 2012. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 384 Whole Genome Analysis of Microlunatus phosphovorus [Vol. 19, polyP hydrolysis,3 using volatile fatty acids such as 2. Materials and Methods acetate as substrates. To date, the features of PAOs have been studied 2.1. Genome sequencing, assembly, and gap closure 6 – 8 mainly in bacterial communities, because few The complete genome sequence of M. phosphovorus such microorganisms have been successfully isolated NM-1T (NBRC 101784T) was determined using a from EBPR sludge. Proteobacteria and/or actinobac- conventional whole genome shotgun strategy, as teria are frequently observed in activated EBPR described previously.18 Shotgun libraries with sludges, and several proteobacteria, e.g. Acinetobacter average insert sizes of 1.5 and 6.0 kb were con- spp. and Lampropedia spp., have been isolated, al- structed in pUC118 vector (TaKaRa, Kyoto, Japan), though their metabolic or morphological characteris- and a fosmid library with an average insert size of tics differ from those typically observed in activated 35 kb was constructed in pCC1FOS vector. Plasmid 3 sludges. Besides these bacterial isolates, an uncultur- and fosmid clones were end-sequenced using dye- able proteobacterium, ‘Candidatus Accumulibacter terminator chemistry on an ABI 3730xl DNA phosphatis’, is regarded as a typical PAO based on its Analyzer (Applied Biosystems, Foster City, CA, USA). 7 PolyP and PHA accumulation properties. Because Raw sequence data corresponding to the 8.6-fold ‘Ca. Accumulibacter phosphatis’ can predominate in coverage were assembled using PHRED/PHRAP/ an EBPR community fed with acetate or propionate, CONSED software.19 Gaps between assembled such sludges have been used for metagenomic and sequences were closed either by primer walking on 9 – 11 metaproteomic analyses. In this way, the molecu- gap-spanning library clones or by the transposon- lar information of polyP accumulating proteobacteria mediated random insertion method on bridging has gradually been accumulating. fosmid clones with a Template Generation System II Less is known about the cellular and molecular Kit (Finnzymes, Vantaa, Finland). features of actinobacterial PAOs. Two species, Microlunatus phosphovorus Tetrasphaera elongata and , 2.2. Gene identification and annotation have been isolated from EBPR-activated sludges as candidate PAOs.12 – 15 These actinobacteria aerobical- Putative non-translated genes were identified using Rfam,20 tRNAscan-SE,21 and ARAGORN22 programs. ly accumulate polyP in their cells, as do proteobacter- 23 ial PAOs. Microlunatus phosphovorus, in particular, To predict protein-coding genes, GLIMMER3 was accumulates substantially more polyP (.10% of cell used. The initial set of open reading frames (ORFs) mass as phosphorus on a dry weight basis) than T. was manually selected from the predictions in com- elongata (,1%) and other proteobacterial PAOs. bination with the similarity search results. Similarity searches against the Uniprot,24 Interpro,25 and Unlike proteobacterial PAOs, these candidates do not 26 12,15,16 HAMAP databases were used for the functional pre- release phosphate when fed with acetate. 27 Instead, glucose and mixed substrates (acetate, casa- diction of ORFs. The KEGG database was used for mino acids, and yeast extract) induce phosphate pathway reconstruction. release in M. phosphovorus and T. elongata, respective- ly. Although PHA synthesis in M. phosphovorus had not 2.3. Data availability been observed for over a decade from its first isola- The nucleotide sequence of M. phosphovorus NM-1T tion, Aker et al.17 recently demonstrated the presence (NBRC 101784T) has been deposited in the INSD of PHA in M. phosphovorus cells using PHA staining and database with an accession number AP012204. The gas chromatography, suggesting the existence of some annotated genome sequence is also available in the metabolic systems for PHA production. genome database DOGAN (http://www.bio.nite.go. In the present study, we determined the complete jp/dogan/project/view/MP1). nucleotide sequence of the M. phosphovorus NM-1T (NBRC 101784T) genome. We put particular focus on (i) polyP synthesis and degradation, (ii) polyP 3. Results and discussion transport, (iii) retention of polyP granules (volutin granules), and (iv) PHA synthesis, which are all 3.1. General information considered to be essential traits of a typical PAO. 3.1.1. Genome overview The genome consisted Very recently, the whole genome sequence of ‘Ca. of a single circular chromosome of 5 683 123 bp Accumulibacter phosphatis’ was made available with an average G þ C content of 67.3% (Fig. 1). No (INSD accession number: CP001715). We discuss plasmid DNA sequence was detected. The chromo- similarities and differences between these two some was predicted to encode 5360 protein-coding genome-sequenced organisms. This report is the first genes, 46 transfer RNA genes, and a set of ribosomal detailed analysis of the whole genome sequences of RNA genes (16S, 23S, and 5S). Of the 5360 predicted PAOs. protein-coding genes, 4887 (91%) were orthologous No. 5] A. Kawakoshi et al. 385 Figure 1. Schematic representation of the circular chromosome of M. phosphovorus. From the periphery toward the centre, circles indicate the scale in Mbp, ORFs predicted on the forward and reverse strands, tRNA genes, rRNA operon, G þ C contents, and GC skew. or had similarity to genes of known function or to 3.1.2. Adaptation in anaerobic conditions hypothetical genes (E-value of ,0.001), and the Microlunatus phosphovorus NM-1T is an obligately remaining 473 (9%) showed no significant similarity aerobic chemoorganotroph but can grow anaerobic- to any registered genes. After manual curation, ally if nitrate is added to the medium as an electron 1999 (37%) genes could be assigned to known bio- acceptor.13 Consistent with this observation, we logical