INTRODUCTION
The phylum‘Verrucomicrobia’(Hedlund et al., 1997) is one of the primary lineages within the domain Bacteria. They represent a distinct lineage within the phylogenetic trees and contain a number of environmental species as well as a small number of cultured species. A number of molecular phyloge- netic approaches and culture-independent studies based on 16S rRNA gene sequences (Hugenholtz et al., 1998; O’Farrell & Janssen, 1999; Rappé &
Giovannoni, 2003) revealed that the members of this phylogenetic group have been detected in a very wide range of quite different habitats within the global ecosystem. Especially, recent results of a phy- logenetic analysis of soil metagenomic libraries (Liles et al., 2003) indicated that many rRNA genes from the bacterial artificial chromosome (BAC) library had substantially affiliated with the bacterial phylum of‘Verrucomicrobia’; all of the sequences were affili- ated with subdivisions that lack cultured representa- tives. At present, these lineages have been informal- ly classified into five subdivisions numbered 1 to 5 (Hugenholtz et al., 1998) and have also been classified into six monophyletic subdivisions numbered 1 to 6 (Vandekerckhove et al., 2000) of which three are rec- ognized in the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity & Holt, 2001) as the families Verrucomicrobiaceae (subdivision 1), Opitutaceae (subdivision 4) and‘Xiphinemato- bacteriaceae’(subdivision 2). Since the six informal monophyletic subdivisions of this phylogenetic group were first proposed, the names of only a few genera such as Akkermansia, Alterococcus, O p i t u t u s , P r o s t h e c o b a c t e r , R u b r i t a l e a a n d Verrucomicrobium (Staley et al., 1976; Schlesner,
1987; Hedlund et al., 1996, 1997; Shieh & Jean; 1998;
Chin et al., 2001; Derrien et al., 2004; Scheuermayer et al., 2006) belonging to subdivisions 1 and 4 have been validly published. Originally, this phylum was known from two genera and five species of prosthe- cate, aerobic heterotrophs isolated from freshwater environments: Verrucomicrobium spinosum, Prosthecobacter fusiformis, P. debontii, P. vanneer- venii and P. dejongeii, although an obligately anaero- bic, heterotrophic genus (Opitutus terrae) from rice paddy field soil has recently been isolated and char- acterized. In addition to the named microorganisms, three taxonomically uncharacterized isolates of
“ultramicrobacteria”from rice paddy field soil with 16S rRNA gene sequences closely related to O. ter- rae have also been described. Alterococcus agarolyti- cus ADT3T, formerly misclassified as a member of the class Gammaproteobacteria, has been proposed to be included in the family Opitutaceae. The class Opitutae, comprising two orders: Puniceicoccales containing the family Puniceicoccaceae and Opitutales containing the family Opitutaceae, was for- mally proposed recently for the classification of sub- division 4 (Choo et al., 2007). However, in spite of wide ecological distribution of representatives of the phylum‘Verrucomicrobia’in nature, owing to rela- tively few pure cultivated and characterized species, the classification of this phylum is still ambiguous and informal. For this reason, for formal classifica- tion of the phylum‘Verrucomicrobia’, it is recom- mended that many verrucomicrobia that thrives in a wide range of terrestrial, aquatic and marine habi- tats should be isolated and taxonomically investigat- ed. Moreover, the remaining subdivisions must be proposed formally by the rank of class and this phy- lum should be also formally proposed.
E-mail: [email protected]
Review Article
Phylogenetic studies on the bacterial phylum‘Verrucomicrobia’
（2011 JSCC Award for Young Scientist）
Jaewoo Yoon
College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea
PHYLOGENETIC STUDIES AND ANALYSIS OF THE CELL WALL COMPONENTS ON THE S T R A I N S O F T H E C L A S S O P I T U T A E (SUBDIVISION 4)
Phylogenetic studies were performed using poly- phasic taxonomic approaches on eleven chemo- heterotrophic bacteria which were isolated from var- ious marine environments. The phylogenetic tree based on the neighbour-joining (NJ) generated com- parisons of the 16S rRNA gene sequences revealed that these isolates were clustered within the family Puniceicoccaceae of the class Opitutae (subdivision 4).
Eleven novel strains represented three independent monophyletic lineages that were distinct from spe- cies with validly published genera with high boot- strap confidence values (Fig. 1). Furthermore, molec- ular (DNA-DNA hybridization test and determina- tion of the DNA G+C content), physiological (acid production from carbohydrates, enzyme activities and basic physiological tests) and biochemical and chemotaxonomic analyses (determination of respira- tory quinone system and cellular fatty acid content and pigments analysis) were investigated on the novel isolates. In addition, increasing concentrations of the β-lactam antibiotic susceptibility tests and amino acid analysis of the cell wall hydrolysates indicated the absence of muramic acid and meso- diaminopimelic acid and the presence of relatively low molar ratios of alanine and glutamate, which suggested that these strains lack an ordinary Gram negative type of peptidoglycan in the cell wall.
Based on the results of phylogenetic analysis and its polyphasic taxonomic evidences, the eleven isolates were considered to represent three novel genera and eight new species belonging to the family Puniceicoccaceae within the class Opitutae (subdivi- sion 4), for which the names Coraliomargarita akaji- mensis gen. nov., sp. nov. (strain 04OKA010-24T; Yoon et al., 2007a), Pelagicoccus mobilis gen. nov., sp.
nov. (strain 02PA-Ca-133T; Yoon et al., 2007b), Pelagicoccus albus sp. nov. (strain YM14-201T; Yoon et al., 2007b), Pelagicoccus litoralis sp. nov. (strains H-MN57T, H-MN48 and MN1-156; Yoon et al., 2007b), Pelagicoccus croceus sp. nov. (strain N5FB36-5T; Yoon et al., 2007d), Cerasicoccus arenae gen. nov., sp.
nov. (strain YM26-026T; Yoon et al., 2007c), Cerasicoccus maritimus sp. nov. (strain YM31-114T; Yoon et al., 2010a) and Cerasicoccus frondicus sp.
nov. (strains YM31-066T and YM31-067; Yoon et al., 2010a) are proposed.
PHYLOGENETIC STUDIES ON THE STRAINS OF THE FAMILY VERRUCOMICROBIACEAE ( S U B D I V I S I O N 1 ) A N D F O R M A L C L A S S I F I C A T I O N O F T H E P H Y L U M
‘VERRUCOMICROBIA’
Nineteen chemoheterotrophic bacterial strains were isolated from various marine environments and organisms, and were subjected to phylogenetic investigations using polyphasic taxonomic methods.
Phylogenetic analysis based on the neighbour-join- ing (NJ) generated comparisons of the 16S rRNA gene sequences indicated that these isolates were affiliated to the family Verrucomicrobiaceae (subdivi- sion 1) within the phylum‘Verrucomicrobia’. Among them, three novel strains had sequences that were related to the genus Rubritalea. Sixteen isolates studied form four different monophyletic clades that were distinct from species with validly published genera with high bootstrap confidence values (Fig.
1). Furthermore, molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (acid production from carbohydrates, enzyme activities and basic physiological tests) and biochemical and chemotaxonomic analyses (determi- nation of respiratory quinone system and cellular fatty acid content and pigments analysis) were investigated on the novel isolates. Some of the novel strains produced carotenoid pigments and squalene.
Analysis of the nearly complete 16S rRNA gene sequences from these strains indicated that the Rubritalea strains formed a robust clade having only limited resolution for this very tight group of spe- cies. However, the interrelationships deduced from the gyrβ gene-based phylogeny showed better reso- lution than those based on their 16S rRNA gene sequences for the differentiation of strains at the species level (Yoon et al., 2010b). The genomic DNA- DNA relatedness values, biochemical and chemotax- onomic data (proportions of cellular fatty acids and menaquinones) also supported the results. On the basis of polyphasic evidences and phylogenetic char- acteristics, it was concluded that these strains should be classified as four novel genera and four- teen new species of the family Verrucomicrobiaceae within the class Verrucomicrobiae (subdivision 1), for which the names Rubritalea spongiae sp. nov. (strain YM21-132T; Yoon et al., 2007e), Rubritalea tangerina sp. nov. (strain YM27-005T; Yoon et al., 2007e), Rubritalea sabuli sp. nov. (strain YM29-052T; Yoon et al., 2008a), Persicirhabdus sediminis gen. nov., sp.
Po1012（DQ302104）T Seawater clone 4（DQ302105）
YM27‑005（AB297806）T YM21‑132（AB297805）T HOact23T（AB277853）
sp. MN1‑1006（AB373024）
YM29‑052（AB353310）T Marine clone SJC3.21（DQ071119）
Polychaete clone A 54（AJ441243）
YM20‑087（AB331886）T YM21‑151（AB331887）
MN1‑741（AB331888）T YM27‑120（AB331889）T
YM26‑010（AB331890）T YM24‑184（AB331891）
YM20‑122 （AB331892）
Elbe river clone DEV005（AJ401105）
Crater lake clone CL500‑85（AF316731）
A5J‑41‑2（AB331893）T A5J‑40（AB331894）
A4T‑83（AB331895）T YM23‑227（AB372852）T 06SJR1‑1（AB372853）T
AK18‑024（AB372854）T 05IJR53‑1（AB372855）T
MN1‑1037（AB372856）T MN1‑1047（AB372857）
ATCC BAA‑835（AY271254）T Human colonic clone HuCa18（AJ408970）
FC4 ATCC 25309（U60015）T FC1 ATCC 27091T（U60012）
FC3i ATCC 700200T（U60014）
FC2 ATCC 700199（U60013）T DSM 4136T（X90515）
Ellin428（AY388649）
Bacterium Ellin502（AY960765）
Bacterium Ellin507（AY960770）
Uncultured bacterium clone DA101（Y07576）
Candidatus （AF217461）
Candidatus （AF217460）
Candidatus （AF217462）
Bacterium Ellin515（AY960778）
Bacterium Ellin518（AY960781）
Bacterium Ellin5102（AY234519）
Freshwateriake clone LD19（AF009974）
PB90‑1 DSM 11246（AJ229235）T ACB90（AJ229246）
PB90‑3（AJ229239）
sp. VeGlc2（X99390）
sp. VeCb1（X99391）
Archaeological wood clone pACH90（AY297806）
sp. strain VeSm13（X99392）
Opitutaceae bacterium TAV1（AY587231）
Uncultured bacterium clone BCf3‑05（AB062814）
Opitutaceae bacterium TAV3（AY587233）
Opitutaceae bacterium TAV4（AY587234）
Opitutaceae bacterium TAV2（AY587232）
BCRC 19135T（AF075271）
YM31‑114（AB372849）T YM26‑026（AB292183）T YM31‑066（AB372850）T YM31‑067（AB372851）
IMCC1545T（DQ539046）
04OKA010‑24（AB266750）T SI‑1234（AB073978）
N5FB36‑5（AB297922）T 02PA‑Ca‑133（AB286015）T YM14‑201（AB286016）T
H‑MN48 MN1‑156
H‑MN57（AB286017）T sp. SA‑9（AY695840）
Contaminated aquifer clone WCHB1‑41（AF050560）
Unidentified marine bacterium clone BD2‑18（AB015546）
Contaminated aquifer clone WCHB1‑25（AF050559）
Anoxic marine sediment clone LD1‑PA26（AY114317）
ATCC 11775T（X80725）
The class The order
The family Verrucomicrobiaceae （Subdivision 1）
The class （Subdivision 2）
Subdivision 3 Subdivision 6
The order The family
The order The family
Subdivision 5
The class （Subdivision 4）
100
100
100 100
100
100 100
100 100
100 100 100
100 100 100
100
100
100
100
100
100
100 100
100
100
100
100 100 99
99
99 97 9398
99 57
99
99
63 95 95
82
92 92
37
66 62
93
87 99
67 54
91
96 82 82
59
46 84
98 98 99 88
99 92
63 92 94
9999
9977
73 0.02
1118 bp
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relative positions of the novel strains described in this study among the currently known and cultivated species of the phylum
‘Verrucomicrobia’. Numbers at the nodes indicate the bootstrap value derived from 1000 replications.
Sequences determined in this study are shown in bold. Bar, 2% sequence divergence.
nov. (strains YM20-087T and YM21-151; Yoon et al., 2008b), Roseibacillus ishigakijimensis gen. nov., sp.
n o v . ( s t r a i n M N1-741T; Y o o n e t a l . , 2008b ) , Roseibacillus ponti sp. nov. (strain YM27-120T; Yoon et al., 2008b), Roseibacillus persicicus sp. nov. (strains YM26-010T, YM24-184 and YM20-122; Yoon et al., 2008b), Luteolibacter pohnpeiensis gen. nov., sp. nov.
(strain A4T-83T; Yoon et al., 2008b), Luteolibacter algae sp. nov. (strains A5J-41-2T and A5J-40; Yoon et al., 2008b), Haloferula harenae gen. nov., sp. nov.
(strain YM23-227T; Yoon et al., 2008c), Haloferula rosea sp. nov. (strain 06SJR1-1T; Yoon et al., 2008c), Haloferula phyci sp. nov. (strain AK18-024T; Yoon et al., 2008c), Haloferula helveola sp. nov. (strain 05IJR53-1T; Yoon et al., 2008c) and Haloferula sargas- sicola sp. nov. (strains MN1-1037T and MN1-1047;
Yoon et al., 2008c) are proposed. With these isolates, emended descriptions of the class Verrucomicrobiae, the order Verrucomicrobiales and the family Verrucomicrobiaceae are presented. Moreover, to establish the formal classification of the phylum
‘V e r r u c o m i c r o b i a’, w e f o r m a l l y p r o p o s e d Verrucomicrobia phy. nov., nom. rev. (Yoon et al., 2010a).
CONCLUSION
We attempted to elucidate the phylogenetic rela- tionships of the thirty novel isolates using polypha- sic taxonomic approaches including 16S rRNA and gyrβ gene sequence analysis, together with molecu- lar, physiological, biochemical and chemotaxonomic analyses to characterize the novel strains. Based on these data, it is proposed that these isolates repre- sent seven novel genera and twenty-two new spe- c i e s w i t h i n t h e p h y l u m‘V e r r u c o m i c r o b i a’. Furthermore, with these novel strains, the formal classification of the phylum‘Verrucomicrobia’is established.
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