Deciphering the Genome of Polyphosphate - DNA Research

12 Pages·2012·0.52 MB·English

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,*
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BiologicalResourceCenter,National Institute ofTechnologyand Evaluation,2-10-49 Nishihara, Shibuya-ku, Tokyo o
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151-0066, Japan1 and Bioproduction Research Institute, National Institute of Advanced Industrial Science and n
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Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan2 a
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*To whom correspondence should be addressed. Tel. þ81 3-6686-2754. Fax. þ81 3-3481-8424. E-mail: fro
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[email protected] h
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Edited by Katsumi Isono ://a
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(Received 25April 2012;accepted 24July2012) d
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Abstract ic.o
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Polyphosphate accumulating organisms (PAOs) belong mostly to Proteobacteria and Actinobacteria and p.c
arequitedivergent.Underaerobicconditions,theyaccumulateintracellularpolyphosphate(polyP),while om
they typically synthesize polyhydroxyalkanoates (PHAs) under anaerobic conditions. Many ecological, /dn
a
physiological,andgenomicanalyseshavebeenperformedwithproteobacterialPAOs,butfewwithactino- re
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bacterial PAOs. In this study, the whole genome sequence of an actinobacterial PAO, Microlunatus phos- e
a
phovorus NM-1T (NBRC 101784T), was determined. The number of genes for polyP metabolism was rch
greater in M. phosphovorus than in other actinobacteria; it possesses genes for four polyP kinases /artic
(ppks), two polyP-dependent glucokinases (ppgks), and three phosphate transporters (pits). In contrast, le
it harbours only a single ppx gene for exopolyphosphatase, although two copies of ppx are generally -ab
s
presentin otheractinobacteria. Furthermore,M. phosphovorus lacksthe phaABC genes forPHA synthesis tra
and the actP gene encoding an acetate/H1 symporter, both of which play crucial roles in anaerobic PHA ct/1
9
accumulation in proteobacterial PAOs. Thus, while the general features of M. phosphovorus regarding /5
aerobic polyP accumulation are similar to those of proteobacterial PAOs, its anaerobic polyP use and /38
3
PHA synthesis appear to be different. /6
2
Key words: Microlunatus phosphovorus; whole genome sequence; polyphosphate accumulating organism; 87
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polyphosphate; polyhydroxyalkanoate 3 b
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1. Introduction biological phosphate removal (EBPR) process, where A
p
they are believed to play a pivotal role in phosphorus ril 2
Economicallyexploitablephosphaterock,themajor removal from the wastewater.3 The EBPR process is 01
9
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
frequentlyfoundinactivatedsludgesintheenhanced polyhydroxyalkanoates (PHAs), at the expense of
#TheAuthor2012.PublishedbyOxfordUniversityPressonbehalfofKazusaDNAResearchInstitute.
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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
mainly in bacterial communities,6–8 because few ThecompletegenomesequenceofM.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 6.0kb were con-
spp. and Lampropedia spp., have been isolated, al- structed in pUC118 vector (TaKaRa, Kyoto, Japan),
though their metabolic or morphologicalcharacteris- and a fosmid library with an average insert size of
tics differ from those typically observed in activated 35kb was constructed in pCC1FOS vector. Plasmid D
sludges.3Besidesthesebacterialisolates,anuncultur- and fosmid clones were end-sequenced using dye- ow
n
able proteobacterium, ‘Candidatus Accumulibacter terminator chemistry on an ABI 3730xl DNA loa
phosphatis’, is regarded as a typical PAO based on its Analyzer (Applied Biosystems, Foster City, CA, USA). de
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PolyP and PHA accumulation properties.7 Because Raw sequence data corresponding to the 8.6-fold fro
‘Ca. Accumulibacter phosphatis’ can predominate in coverage were assembled using PHRED/PHRAP/ m h
an EBPR community fed with acetate or propionate, CONSED software.19 Gaps between assembled ttp
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smuecthapsrluodtegoems ihcaavneablyeseens.9u–s1e1dInfotrhimswetaayg,ethneommiocleacnud- sgeaqpu-sepnacnensinwgerleibcrlaorsyedcloeintehserobrybpyritmheertwraanlkspinogsoonn- ://aca
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lar information of polyP accumulating proteobacteria mediated random insertion method on bridging em
has gradually been accumulating. fosmid clones with a Template Generation System II ic
.o
Less is known about the cellular and molecular Kit (Finnzymes, Vantaa, Finland). up
features of actinobacterial PAOs. Two species, .c
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Microlunatus phosphovorus and Tetrasphaera elongata, 2.2. Gene identification and annotation /d
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have been isolated from EBPR-activated sludges as a
candidate PAOs.12–15 These actinobacteria aerobical- Putative non-translated genes were identified using res
lyaccumulate polyP in theircells, as doproteobacter- RTofampr,2e0dictRtNpAroscteainn--ScEo,2d1inagndgeAnResA,GGOLRIMN2M2EpRr3o2g3rawmass. earch
ial PAOs. Microlunatus phosphovorus, in particular, /a
accumulates substantially more polyP (.10% of cell used. The initial set of open reading frames (ORFs) rtic
was manually selected from the predictions in com- le
mass as phosphorus on a dry weight basis) than T. -a
bination with the similarity search results. Similarity b
elongata (,1%) and other proteobacterial PAOs. s
searches against the Uniprot,24 Interpro,25 and tra
Unlike proteobacterial PAOs, these candidates do not c
release phosphate when fed with acetate.12,15,16 HAMAP26databaseswereusedforthefunctionalpre- t/19
diction of ORFs. The KEGG27 database was used for /5
Instead, glucose and mixed substrates (acetate, casa- /3
pathway reconstruction. 8
mino acids, and yeast extract) induce phosphate 3
/6
releaseinM.phosphovorusandT.elongata,respective- 2
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ly.AlthoughPHA synthesisinM.phosphovorushadnot 2.3. Data availability 72
3
been observed for over a decade from its first isola- ThenucleotidesequenceofM.phosphovorusNM-1T b
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tion,Akeretal.17recentlydemonstratedthepresence (NBRC 101784T) has been deposited in the INSD gu
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ofPHAinM.phosphovoruscellsusingPHAstainingand database with an accession number AP012204. The s
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gaschromatography,suggestingtheexistenceofsome annotated genome sequence is also available in the n
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metabolic systems for PHA production. genome database DOGAN (http://www.bio.nite.go. 6
A
In the present study, we determined the complete jp/dogan/project/view/MP1). p
nucleotide sequence of the M. phosphovorus NM-1T ril 2
0
(NBRC 101784T) genome. We put particular focus 19
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 5683123bp
Accumulibacter phosphatis’ was made available with an average GþC 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 RNAgenes(16S,23S,and5S).Ofthe5360predicted
PAOs. protein-coding genes, 4887 (91%) were orthologous
No. 5] A. Kawakoshi et al. 385
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Figure1. SchematicrepresentationofthecircularchromosomeofM.phosphovorus.Fromtheperipherytowardthecentre,circlesindicate trac
thescaleinMbp,ORFspredictedontheforwardandreversestrands,tRNAgenes,rRNAoperon,GþCcontents,andGCskew. t/1
9
/5
/3
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or had similarity to genes of known function or to 3.1.2. Adaptation in anaerobic conditions 3
/6
hypothetical genes (E-value of ,0.001), and the Microlunatus phosphovorus NM-1T is an obligately 28
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remaining 473 (9%) showed no significant similarity aerobic chemoorganotroph but can grow anaerobic- 2
3
to any registered genes. After manual curation, ally if nitrate is added to the medium as an electron by
1999 (37%) genes could be assigned to known bio- acceptor.13 Consistent with this observation, we gu
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logical roles. Microlunatus phosphovorus harboured found agene cluster that putativelyencodes subunits st o
only a single rRNA operon, a significant finding given of the membrane-bound respiratory nitrate reduc- n
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its relatively large genome. The general features of tase, NarG (MLP_46640), NarH (MLP_46650), NarJ 6 A
tthhoeseM.opfho1sp4hovootrhuesrgeancotimnoebaacrteerciaolmpspareecdieswitihn (NMarLKP-_t4y6pe660),naitnradteN/naritIri(tMeLP_4tr6a6n7sp0o),rtleinrkedgteonea pril 2
0
1
Supplementary Table S1. In this comparison, target (MLP_46680). In addition, we found a gene that 9
genomes were selected to represent each taxonomic encodesanotherNarK-typenitrate/nitritetransporter
rank from the genus Microlunatus stepwise to the (MLP_35250)locatedthedownstreamofputativeas-
class Actinobacteria. Supplementary Table S2 shows similatory nitrite reductase genes (MLP_35260 and
thedistributionofORFsinCOGfunctionalcategories. MLP_35270). These components together may con-
No significant overrepresentation or underrepre- stitute a system for nitrate respiration in anaerobic
sentation of any particular functional category in conditions as well as for nitrogen assimilation. Under
M. phosphovorus was observed except that a higher anaerobic conditions, M. phosphovorus NM-1T was
percentage of ORFs was categorised as ‘function reportedtogenerateacetatefromglucose,suggesting
unknown’ in M. phosphovorus than in other the presence of an acetate fermentation system.28 In
actinobacteria. support of previous experimental data,28 we found
386 Whole Genome Analysis of Microlunatus phosphovorus [Vol. 19,
putative genes, pta-ackA (MLP_01330 and variety of bacterial species.36 Even though the reac-
MLP_01320), encoding phosphate acetyltransferase tion catalysed by PPK is reversible, PPK1 favours
and acetate kinase, which usually ferment acetate in polyP synthesis with nucleoside triphosphates as
bacteria. Furthermore, putative pflBA genes phosphate donors, and thus PPK1 is recognized to
(MLP_33410 and MLP_33420), which are necessary play a principal role in polyP accumulation. On the
for the synthesis and activation of pyruvate formate other hand, PAP favours polyP hydrolysis. The PPK2
lyase,acentralenzymeinanaerobicglucosemetabol- subtype also catalyses polyP hydrolysis, but the dom-
ism, were found in the M. phosphovorus genome. inance of either kinase or phosphatase activity varies
Because pflBA genes are present in few lineages of in different actinobacterial species; Corynebacterium
actinobacteria,andbecauseanIS-likesequence,apu- glutamicum PPK2 is a polyP kinase and
tative transposase gene flanked by 17-bp inverted Mycobacterium tuberculosis PPK2 is a polyP phosphat- D
repeat sequences, was present just upstream of the ase.37–39 ow
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pflBA genes, pflBA could have been acquired by hori- Four putative PPK genes were identified in the lo
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zontal gene transfer. M. phosphovorus genome; a single ppk1 (MLP_47700) de
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and three ppk2 (MLP_05750, MLP_50300, and fro
3.1.3. Substrates for growth Metabolic pathways MLP_23310)homologues.Thenumberofppkhomo- m
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reconstructed in this study were roughly in accord- logues in M. phosphovorus was relatively large; usually ttp
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apnhocveowruitshNpMre-v1ioTu(sSsutupdpileemsoenntnaurtyrieTnabtlueseS3in).M13.,2p9h–o3s1- 1ge–n4ompepk (hToambleolog1u)e.s Beaxissetdinonan tahcetinosbimacitlaerriitayl ://ac
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However, while lactose and malate were predicted to of deduced amino acid sequences, one of the e
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be used as nutrients based on the genome informa- M. phosphovorus PPK2s (MLP_05750) was of the ic
tion, the growth of M. phosphovorus NM-1T on these C. glutamicum type (63% identity), whereas the .ou
p
nutrients has not been observed. On the other other (MLP_50300) was of the Myc. tuberculosis .c
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hand, while growth has been experimentally shown type (78% identity; Fig. 2a). The third ppk2 homo- /d
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on mannose, galactose, N-acetyl-D-glucosamine, logue (MLP_23310) was relatively similar to both a
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sorbose, salicin, p-arbutin, dulcitol, and adonitol, ppk2 and pap, but was located in a distinct cluster of s
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metabolic pathways for the use of these compounds undetermined function (Fig. 2a). arc
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were either missing or incomplete. Other substrates The presence of multiple PPK genes in /a
that have not yet been experimentally investigated, M. phosphovorus may have favoured the high polyP rtic
le
such as formate and butyrate, might also be used turnover rate and accumulating ability, although -a
b
based on the predicted pathways. Butyrate metabol- whichofthefourPPKhomologuesarepolyP-synthetic s
tra
ism may be advantageous for PAOs because butyrate, ordegradativehasyettobeclearlydistinguished.The c
aswellasacetate,couldbeamajorfattyacidavailable possible importance of multiple PPK genes was t/19
in sewage water, and butyrate was consumed when supported by the genome information of ‘Ca. /5/3
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nitrateisaddedunderanaerobicconditionsinanacti- Accumulibacter phosphatis’. ‘Ca. Accumulibacter 3
vated sludge sample.32 phosphatis’ harbours multiple putative PPK genes, a /62
8
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ppk1, four ppk2, and a pap, while proteobacterial 2
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species usually contain onlyone or two ppk subtypes. b
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3.2. Predicted features as a PAO g
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3.2.1. PolyP metabolism As described above, s
M. phosphovorus NM-1T aerobically accumulates 3.2.1.2. Exopolyphosphatase t on
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polyP in its cells, and phosphorus can exceed 10% Exopolyphosphatase (PPX) mediates the hydrolysis 6
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of the cell mass on a dry weight basis.13 In addition, of the terminal phosphate of polyP.36 Two types of p
the rate of phosphorus release under anaerobic PPX, PPX1 and PPX2, are present in a wide variety of ril 2
0
conditions is significantly higher in M. phosphovorus bacteria and archaea.36 Although most actinobacter- 19
NM-1T than in any other isolated PAO candi- ial species harbour both types of PPX, only the
date.12,28,33–35 The following sections describe the ppx2 homologue (MLP_44770) was found in the
gene products that were implicated in the accumula- M. phosphovorus genome (Fig. 2b). Propionibacterium
tion, degradation, and high turnover rate of polyP in acnes, which also belongs to the family
M. phosphovorus. Propionibacteriaceae, does not harbour ppx1 either.
In C. glutamicum, which was recently shown to form
3.2.1.1. PolyP kinase PolyP kinase (PPK) cata- volutin granules, mutational analysis showed that
lyses the transfer of phosphate between nucleoside the amount of polyP in the cells increased when one
phosphates and polyP. There are three main subtypes ofthese geneswas mutated.40IncontrasttoM. phos-
of PPK, PPK1, PPK2, and polyP-dependent AMP phos- phovorus,‘Ca.Accumulibacterphosphatis’,theproteo-
photransferase (PAP), and they are present in a wide bacterialPAO,hastwoppxhomologuesinitsgenome.
No. 5] A. Kawakoshi et al. 387
Table1. NumberofputativegenesrelatedtopolyPmetabolismin suggesting the importance of polyP as an energy
actinobacteria source for M. phosphovorus.
In the present study, three genes putatively encod-
ppk ppx ppgK pit pstSCAB
ingglucokinasewereidentifiedintheM.phosphovorus
Propionibacteriaceae
genome; one was putative ATP-dependent glk
Microlunatusphosphovorus 4 1 2 3 1
(MLP_41670) and another (MLP_05430) was the
Propionibacteriumacnes 3 1 1 1 1 ppgK reported previously.48 The third gene
Propionibacterineae (MLP_26610) was inferred to also be ppgK by
Nocardioidessp.JS614 3 2 1 1 2 molecular phylogenetic analysis (Fig. 2c). Internal
Actinomycetales deletions characteristic of PPGKs, as well as other
Catenulisporaacidiphila 3 2 0 2 2 functional motifs including the possible polyP- D
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binding site, were observed in the deduced amino w
Corynebacteriumglutamicum 3 2 1 1 1 n
acid sequence of MLP_26610 (Supplementary lo
Frankiaalni 3 2 1 1 1 Fig. S1), suggesting that MLP_26610 is another ppgK ade
Kineococcusradiotolerans 3 2 1 2 1 orthologue (hereafter MLP_05430 and MLP_26610 d fro
Kocuriarhizophila 2 2 1 2 1 are designated ppgK1 and ppgK2, respectively). This m
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Mycobacteriumsmegmatis 3 2 1 1 1 is the first report of two ppgK orthologues being ttp
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Rhodococcuserythropolis 4 2 1 2 2 presentinasingleorganism(Table1).Inthepresence ://a
Streptomycesavermitilis 2 3 1 2 1 of glucose, M. phosphovorus may use the dual ppgKs ca
d
for efficient glycolysis by consuming intracellular e
Actinobacteridae m
polyP, which isthen followed byacetate fermentation ic
Bifidobacteriumlongum 2 2 1 1 1 .o
(seethe ‘Adaptation in anaerobic conditions’ section) u
Actinobacteria(class) to adapt to anaerobic environments. This notion p.c
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Acidimicrobiumferrooxidans 2 1 0 0 0 m
agrees well with the observation that adding glucose /d
Cryptobacteriumcurtum 1 1 0 1 0 to a pure culture of M. phosphovorus NM-1T under na
Rubrobacterxylanophilus 1 0 1 0 1 anaerobic conditions resulted in a rapid release of Pi res
e
Accession numbers of genomic sequences in INSD: from the cells.12,16 arc
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Propionibacterium acnes, AE017283; Nocardioides sp. JS614, /a
CP000509; Catenulispora acidiphila, CP001700; 3.2.2. Phosphate transport systems For rapid rtic
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Corynebacterium glutamicum, BX927147; Frankia alni, polyP metabolism, M. phosphovorus must have -a
b
CT573213;Kineococcusradiotolerans,CP000750;Kocuriarhi- efficient phosphate uptake and release. In bacteria, stra
zophila, AP009152; Mycobacterium smegmatis, CP000480; phosphate uptake commonly occurs via the phos- c
Rhodococcus erythropolis, AP008957; Streptomyces avermitilis, phate-specifictransport (Pst)andphosphateinorgan- t/19
BA000030; Bifidobacterium longum, CP000605; ic transport (Pit) systems.49 The Pst is an ATP-binding /5/3
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Acidimicrobium ferrooxidans, CP001631; Cryptobacterium cassette (ABC) transporter, encoded by the gene 3/6
curtum,CP001682;Rubrobacterxylanophilus,CP000386. clusterpstSCAB,foractivePit,whilethePitisasympor- 28
7
ter of divalent metal-chelated phosphate (MeHPO ) 2
4 3
and Hþ. In some Gram-negative bacteria, another b
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ABC transporter, PhnCDE, has been identified.50–52 g
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3.2.1.3. PolyP-dependent Recently, the PhnCDE transport system was also es
glucokinase Glucokinase is a key identified in an actinobacterium, Mycobacterium t on
enzyme that catalyses phosphorylation of glucose, smegmatis,53 although the system is not commonly 06
A
the first reaction of glycolysis. Glucokinase is widely present in this phylum. p
present in both prokaryotes and eukaryotes.41 In A putative pstSCAB (MLP_47720, MLP_47730, ril 2
0
almost all organisms, glucokinase uses ATP as the MLP_47740, and MLP_47750) and three pits 19
sole phosphoryl donor. In some actinobacterial (MLP_00530, MLP_29830, and MLP_51060) were
species, however, a paralogue of glucokinase, polyP- identified in the M. phosphovorus genome. Notably,
dependent glucokinase (PPGK), has been identified the number of pit genes in M. phosphovorus was
which uses polyP as the phosphoryl donor as well as among the largest known in an actinobacterial
ATP.42–46 In C. glutamicum, western blot analysis species(Table1).Basedonthemolecularphylogenet-
showed that putative PPGK was localized in the ic analysis, MLP_29830 and MLP_51060 were most
volutin granules, suggesting that PPGK uses polyP homologous to those in actinobacteria (Fig. 2d). On
fromthosegranules.47APPGKhasalreadybeeniden- the other hand, MLP_00530 seemed not to be an
tified in M. phosphovorus NM-1T.48 That report actinobacterialgenebutrather wassimilartoproteo-
showed that the PPGK was strictly polyP- bacterial genes(Fig.2d)withamaximum amino acid
specific, unlike other actinobacterial PPGKs, identity of 48%. This might suggest that MLP_00530
388 Whole Genome Analysis of Microlunatus phosphovorus [Vol. 19,
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Figure2. Molecularphylogeneticanalysesofthe(A)polyPkinasefamily(PPK1,PPK2andPAP)inbacteria,(B)exopolyphosphatasefamily 0
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(PPX1andPPX2)inactinobacteria,(C)glucokinasefamily(PPGKandGK)inprokaryotes,withsomeeukaryotes,and(D)phosphate A
tarcaidnsspeoqruteernsce(Psitd)eidnucaectdinforobmactneurcialeaontiddeprsoetqeuoebnaccetserwiae.rTereuessedwaesreocpoenrasttirouncateldtauxosinnogmthiceudnisittsan(OceTUmsa)tfroixrmtheethaondalyinseCs.luAsltlatloXp.oAlmogiineos pril 2
0
agreedwiththoseofmaximumparsimonyanalysesinPHYLIP.Thenumbersoninteriornodesarebootstrappercentages.Singleand 1
9
double asterisks indicate OTUs in which the polyP-synthetic or degradative reaction is dominant, respectively, and the abbreviation
‘U.C.’indicatesaclusterforwhichthefunctionhasyettobecharacterised.
was obtained from another phylum, such as mandatory in the group; pit in Myc. smegmatis was
Proteobacteria, although an upstream genewith a pu- dispensable, with the Pst and Phn systems suggested
tative regulatory function (Pfam: PF01865), which is to compensate for the function of Pit,54 and some
commonly associated with proteobacterial Pit genes, actinobacteria do not harbour pit in their genomes
could not be found in the case of MLP_00530. (Table 1). The reversible Pi transport mediated by Pit
Thus far, the Pit system has not been much studied without consuming ATP may be favourable to cope
inGram-positivebacteria.Rather,itappearsnottobe with drastic changes in intracellular Pi concentration
No. 5] A. Kawakoshi et al. 389
due to rapid polyP synthesis and degradation in PAO. M. phosphovorus speA to non-actinobacterial genes
The possible importance for PAOs of multiple pit are modest, with amino acid identities less than
genes is supported by the fact that the genome of 33%, obscuring the exact origin of this gene. In con-
‘Ca. Accumulibacter phosphatis’ harbours four pit trast, the homologue of speE, which is usually
homologues,morethanotherproteobacterialspecies. present in actinobacteria, was not identified in the
M. phosphovorus genome.
3.2.3. Retention of volutin granules To accumu- Busse and Schumann57 reported that spermidine
late a large amount of intracellular polyP, polyP must and spermine were the major polyamines in the
be stably maintained as volutin granules. Although cells of M. phosphovorus NM-1T, while only a trace
molecular mechanisms of volutin granule formation amount of putrescine was found. This result contra-
have not been clarified in detail, polyamines were dicts the prediction from the genomic data, i.e. D
o
suggested to play a role in polyP accumulation M. phosphovorus can synthesize only putrescine w
n
in Escherichia coli.55 Polyamines mainly consist of among the three polyamines, as far as the currently lo
a
putrescine, spermidine, and spermine in eukaryotic recognized metabolic pathway (Fig. 3) is taken into de
d
cells, whereas spermine is contained in only a few account. Perhaps spermidine and/or spermine are fro
prokaryotic species, including some actinobacteria.56 synthesized via unknown synthases or taken up m
h
Polyamines are aliphatic amines that are highly- actively by polyamine transporters. M. phosphovorus ttp
s
charged cations under physiological conditions. They harbours eight putative genes whose deduced ://a
are generallyrecognized as essential forthemainten- amino acid sequences contain the amino acid/ c
a
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ance of cell growth and macromolecular biosynthesis polyamine transporter I motif (InterPro ID: e
m
by interacting with nucleic acids, proteins, and mem- IPR002293). These transporters may be related to ic
branes. In addition, Motomura et al.55 demonstrated the stabilization of volutin granules, although their .ou
p
thatintracellularpolyPincreasedafteraddingspermi- substrate specificities could not be clearly assigned. .c
o
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dineandputrescinetoE.colicellsinwhichpolyamine /d
synthesis genes had been disrupted. They further 3.2.4. PHA synthesis Inadditionto aerobicpolyP na
re
demonstrated in vitro that adding polyamines to accumulation, anaerobic PHA production is a recog- s
e
volutingranule-containingsolutionsincreasedthere- nizedfeatureofPAOs.3Recently,Akeretal.17reported arc
tention timeofthe granulesandthatspermidinewas the detection in M. phosphovorus NM-1T cells of two h/a
the best polyamine for stabilizing the granules. PHA species,polyhydroxybutyrateandpolyhydroxyva- rtic
le
Polyamines are synthesized from L-arginine or lerate, by Sudan Black B- and Safranin O-staining and -a
b
Lo-xoyrlnaistehi(nSepevAia)raenadctaiognmsactaintaelyusreedohbyydarrogliansiene(SdpeecBa)robr- gPaHsAcphrroodmuacttoiognraipnhMym. pehtohsopdhso.vHorouwseavpepr,etahresstoystbeemdiof-f strac
ornithine decarboxylase (SpeC) (Fig. 3). The synthe- ferentfromthatproposedinproteobacterialPAOs;M. t/19
sized putrescine is then converted to spermidine and phosphovorus apparently produces PHA under aerobic /5/3
8
spermine in the reactions catalysed by spermidine conditions,whereasproteobacterialPAOsarebelieved 3
/6
synthase (SpeE) and spermine synthase, respectively, to synthesize PHAs anaerobically. In proteobacterial 2
8
although spermine synthase has not been found in PAOs, PHA is produced using glycogen/glucose or 72
3
prokaryotes thus far.56 In the M. phosphovorus volatile fatty acids such as acetate as substrates, and b
y
genome, putative speA (MLP_07520) and speB theuptakeoftheacetateisconductedviaActP,apro- g
u
(MLP_15750) were identified but did not form a teobacteria-specific acetate/Hþ symporter. Acetate es
t o
gene cluster. Although speB is conserved among acti- uptakethroughActPishypothesizedtobecounterba- n
0
nobacteria, speA or its homologues are rarely identi- lanced by Pi release via Pit using the proton motive 6
A
fied in actinobacteria. In addition, similarities of force.58,59 The intermediate acetyl-CoA is then p
converted to PHAs by acetyl-CoA acetyltransferase ril 2
0
(PhaA), acetoacetyl-CoA reductase (PhaB), and PHA- 19
synthase (PhaC) (Fig. 4).60 This model was further
supported in a proteobacterial PAO, ‘Ca.
Accumulibacter phosphatis’, by metagenomic and
metaproteomic analyses.9,11 In contrast, neither actP
nor phaABC exists in most actinobacteria, and these
genes were not found in the M. phosphovorus
genome either.
Figure 3. Polyamine metabolism and related enzymes. Shadows In addition to the Pha system, other pathways
indicate enzymes for which putative genes were predicted in derived from the b-oxidation pathway proposed in
the M. phosphovorus genome. Polyamine species that were
mainly detected in M. phosphovorus cells57 are indicated by E. coli and Pseudomonas putida could synthesize PHA
asterisks. (Fig. 4).61–63 In E. coli, the gene cluster yfcYX is
390 Whole Genome Analysis of Microlunatus phosphovorus [Vol. 19,
M. phosphovorus takes up Pi through a PstSCAB and
multiple Pits. PhnCDE rarely exists in non-proteobac-
teria and is absent in M. phosphovorus. For Pi uptake
via Pits, a proton motive force is required. In ‘Ca.
Accumulibacter phosphatis’, the electron transport
chain of aerobic respiration was proposed for that
function11 and may be the primary source of the
proton motive force in aerobic M. phosphovorus, as
well. The ingested intracellular Pi isthen polymerized
into polyP by some of the multiple PPK(s) and stored
as volutin granules. In M. phosphovorus, PHA may be D
o
synthesized through the b-oxidation pathway, w
n
whereas proteobacteria degrade PHA and utilize it as lo
a
an energy source under the aerobic conditions. de
d
Under anaerobic conditions, polyP is degraded by fro
otherPPK(s),aPPX2andtwoPPGKs.Glucose-6-phos- m
h
phate generatedbyPPGKs ispossiblyfedinto glycoly- ttp
s
sis followed by acetate fermentation, and the NADH ://a
producedintheprocesscanbeusedfornitraterespir- c
a
d
ation and nitrogen assimilation. Because PPGK has e
m
been identified only in actinobacteria thus far, this ic
.o
glycolytic pathway coupled with polyP consumption u
p
Figure4. TwoindependentpathwaysforPHAsynthesisthrough(A) would be unique within this phylum. The release of .co
thePhaABCsystem,and(B)theb-oxidationpathway.Enzymesfor Pi through Pit symporters generates a proton motive m/d
which putative genes were predicted in the M. phosphovorus n
force. While the proton motive force is thought to a
genomeareshadowed. re
beusedforacetateuptakeviaActPin proteobacterial s
e
relatedtothepathway;theencodedYfcXwasdemon- PAOs,thismodelcannotbeappliedtonon-proteobac- arc
strated to be a multifunctional protein with enoyl- teria, because ActP is present almost exclusively in h/a
CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, proteobacteria. Instead, the proton motive force may rtic
le
and putative 3-hydroxyacyl-CoA epimerase activities, be used, at least in part, for the symport of nitrate -a
b
andYfcYtobeab-ketothiolase.InP.putida,(R)-specif- by NarK-type transporters in M. phosphovorus. s
ic enoyl-CoA hydrolase (PhaJ) is involved in the The mechanism of anaerobic polyP utilization in trac
system. Both YfcX and PhaJ result in the synthesis of M. phosphovorus thus appears to be substantially t/19
(R)-3-hydroxyacyl-CoA, the monomer unit of PHAs. different from that in proteobacterial PAOs, in con- /5/3
In the M. phosphovorus genome, homologues of trast to the general similarities seen in aerobic polyP 83
/6
yfcYX (MLP_23080 and MLP_23090) and phaJ accumulation. In proteobacteria, the polyP degrad- 2
8
(MLP_12780) were identified, suggesting that these ation system is thought to be tightly linked to 72
3
genes might produce PHA in M. phosphovorus rather acetate uptake and the synthesis of PHA as an b
y
than the PhaABC system proposed in proteobacterial energy reservoir. The system in M. phosphovorus, g
u
e
PAOs. There remains an unresolved issue; both however, does not appear to be directly linked to s
pathways require PhaC for the final polymerization the synthesis of PHA, but may couple with the t on
0
reaction, but the gene is not present in the carbon and energy metabolism necessary for a 6
A
M. phosphovorus genome. Novel unidentified PHA minimal level of growth under anaerobic conditions. p
synthase(s) might exist in M. phosphovorus that This might be another reason why M. phosphovorus ril 2
0
enable it to synthesize PHAs via a pathway independ- can accumulate much larger amounts of polyP 19
ent of polyP degradation and distinct from that con- under the alternating aerobic/anaerobic conditions
ventionally proposed in proteobacterial PAOs. in the EBPR process.
3.2.5. A possible model for an actinobacterial
PAO Based on the genome analysis as 3.3. Future perspectives
described in this report and previous experimental In the present study, we found genetic features in
observations, possible pathways of M. phosphovorus the M. phosphovorus genome that could allow it to
that may represent the features of an actinobacterial express phenotypic characteristics of a PAO. Some
PAO were summarized in Fig. 5 (upper panels) to- of these features, such as the high multiplicity of
gether with a model proposed in proteobacterial ppk and pit genes, were commonly seen in the
species(lowerpanels).4,9,11Underaerobicconditions, genome of a proteobacterial PAO candidate, ‘Ca.
No. 5] A. Kawakoshi et al. 391
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
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a
d
e
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ic
.o
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.c
o
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e
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rc
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/a
rtic
le
-a
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tra
c
t/1
Figure5. SchematicrepresentationofpossiblepathwaysforpolyPandPHAmetabolismsunder(A)aerobicand(B)anaerobicconditions. 9/5
TheupperpanelsineachfigurerepresentmodelsproposedinthisstudyforM.phosphovorus,whiletheloweronesrepresentmodels /3
8
recognizedinproteobacterialspecies.Notallintermediatereactionsareshownindetail. 3
/6
2
8
Accumulibacter phosphatis’, supporting the import- listed in Table 1. From the aspect of genome 72
3
ance of these genes for polyP metabolisms. On the evolution, a possible ancestral genetic locus for b
y
otherhand,someotherfeatures,suchasthepresence phosphate metabolism can be seen in the genome g
u
e
of duplicated ppgk genes and the unique absence of of Gemmatimonas aurantiaca (DOGAN database: s
onetypeofppxgene,werespecifictotheM.phospho- http://www.bio.nite.go.jp/dogan/project/view/GA1, t on
0
vorus genome. Yet to be elucidated, however, is accession number in INSD: AP009153) which was 6
whether other factors, such as expression levels and also isolated from an EBPR-activated sludge.64 In Ap
activities of these gene products or their differential this organism, all predicted genes related to phos- ril 2
0
regulation, would also affect the PAO phenotype. phate metabolism (a pstSCAB, a ppx, a ppk, a pit, 19
Genes possibly related to polyP accumulation were and five regulator genes) occur as a single gene
widely dispersed across the genome of M. phospho- cluster. Provided that the gene cluster represents an
vorus, and molecular phylogenetic analyses suggested ancestral trait, PAOs may have evolved convergently
that some had exogenous origins. This tendency was in a variety of taxa via complex recombinational
also seen in the ‘Ca. Accumulibacter phosphatis’ events including duplication, deletion, and horizontal
genome (data not shown). In support of the high gene transfer that occurred independently in each
plasticity of M. phosphovorus genome, the number lineage.
of genes whose predicted protein product had Recently, five species in the genus Microlunatus
at least one Pfam domain related to transposase, have newly been isolated; none accumulates
integrase, or recombinase was highest in M. phospho- polyP.29–31,65,66 Genome data for these related
vorus (99 genes) among the actinobacterial species species and comparative genomics approaches
392 Whole Genome Analysis of Microlunatus phosphovorus [Vol. 19,
would provide a clearer picture of the genetic back- and aerobic phases of a bioreactor operating for
ground characteristic of PAOs. enhanced biological phosphorus removal, Environ.
Microbiol.,11, 3029–44.
11. Garc´ıa-Mart´ın, H., Ivanova, N., Kunin, V., et al. 2006,
Acknowledgements: The authors thank Drs
Metagenomic analysis of two enhanced biological
Tomohiko Tamura, Misa Otoguro and Ken-ichiro
phosphorus removal (EBPR) sludge communities, Nat.
Suzuki for their useful discussions and the help in
Biotechnol., 24, 1263–9.
cell culture.
12. Nakamura, K., Masuda, K. and Mikami, E. 1991,
Isolationofanewtypeofpolyphosphateaccumulating
Supplementary data: Supplementary Data are
bacterium and its phosphate removal characteristics,
available at www.dnaresearch.oxfordjournals.org. J.Ferment. Bioeng., 4,258–63.
13. Nakamura,K.,Hiraishi,A.,Yoshimi,Y.,Kawaharasaki,M., D
o
Masuda, K. and Kamagata, Y. 1995, Microlunatus w
Funding phosphovorus gen. nov., sp. nov., a new gram-positive nlo
a
polyphosphate-accumulating bacterium isolated from d
e
MiTnhisitsrywoofrEkcownoasmys,uTprpaodreteadndbIyndugrsatrnytsoffJraopman.the 14. aHcatnivaadtead, Ss.l,udLgiue,,IWnt..T.J,.SSyhsitn.tBaanci,terTi.o,l.K,a4m5,ag1a7ta–,22Y.. and d from
Nakamura, K. 2002, Tetrasphaera elongata sp. nov., h
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