Download Phylogenetic studies on the bacterial phylum`Verrucomicrobia`

Microbiol. Cult. Coll. 27（2）
：61 ─ 65, 2011
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
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 phylogenetic 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 phylogenetic 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 affiliated with subdivisions that lack cultured representatives. At present, these lineages have been informally 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 recognized in the second edition of Bergey’
s Manual of
Systematic Bacteriology (Garrity & Holt, 2001) as the
families Verrucomicrobiaceae (subdivision 1),
Opitutaceae (subdivision 4) and‘Xiphinematobacteriaceae’
(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,
Opitutus, Prosthecobacter, Rubritalea and
Verrucomicrobium (Staley et al., 1976; Schlesner,
E-mail: [email protected]
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 prosthecate, aerobic heterotrophs isolated from freshwater
environments: Verrucomicrobium spinosum,
Prosthecobacter fusiformis, P. debontii, P. vanneervenii and P. dejongeii, although an obligately anaerobic, heterotrophic genus (Opitutus terrae) from rice
paddy field soil has recently been isolated and characterized. 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. terrae have also been described. Alterococcus agarolyticus 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 formally proposed recently for the classification of subdivision 4 (Choo et al., 2007). However, in spite of
wide ecological distribution of representatives of the
phylum
‘Verrucomicrobia’
in nature, owing to relatively few pure cultivated and characterized species,
the classification of this phylum is still ambiguous
and informal. For this reason, for formal classification of the phylum‘Verrucomicrobia’
, it is recommended that many verrucomicrobia that thrives in a
wide range of terrestrial, aquatic and marine habitats should be isolated and taxonomically investigated. Moreover, the remaining subdivisions must be
proposed formally by the rank of class and this phylum should be also formally proposed.
─ 61 ─
Phylogenetic studies on the bacterial phylum‘Verrucomicrobia ’
PHYLOGENETIC STUDIES AND ANALYSIS OF
THE CELL WALL COMPONENTS ON THE
STRAINS OF THE CLASS OPITUTAE
(SUBDIVISION 4)
Phylogenetic studies were performed using polyphasic taxonomic approaches on eleven chemoheterotrophic bacteria which were isolated from various marine environments. The phylogenetic tree
based on the neighbour-joining (NJ) generated comparisons 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 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 (determination of respiratory 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 mesodiaminopimelic 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 (subdivision 4), for which the names Coraliomargarita akajimensis gen. nov., sp. nov. (strain 04OKA010-24T ;
Yoon et al., 2007a), Pelagicoccus mobilis gen. nov., sp.
nov. (strain 02PA-Ca-133 T ; 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.
n o v . ( s t r a i n Y M26-026 T ; Y o o n e t a l . , 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.
Jaewoo Yoon
PHYLOGENETIC STUDIES ON THE STRAINS
OF THE FAMILY VERRUCOMICROBIACEAE
(SUBDIVISION 1) AND FORMAL
CLASSIFICATION OF THE PHYLUM
‘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-joining (NJ) generated comparisons of the 16S rRNA
gene sequences indicated that these isolates were
affiliated to the family Verrucomicrobiaceae (subdivision 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 (determination 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 species. However, the interrelationships deduced from
the gyrβ gene-based phylogeny showed better resolution than those based on their 16S rRNA gene
sequences for the differentiation of strains at the
species level (Yoon et al., 2010b). The genomic DNADNA relatedness values, biochemical and chemotaxonomic data (proportions of cellular fatty acids and
menaquinones) also supported the results. On the
basis of polyphasic evidences and phylogenetic characteristics, it was concluded that these strains
should be classified as four novel genera and fourteen 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 YM27-005 T ; Yoon et al., 2007e),
Rubritalea sabuli sp. nov. (strain YM29-052T; Yoon et
al., 2008a), Persicirhabdus sediminis gen. nov., sp.
─ 62 ─
Microbiol. Cult. Coll. Dec. 2011
Vol. 27, No. 2
0.02
1118 bp
82
95
95
63
100
87
100
100
T
Po1012（DQ302104）
Seawater clone 4（DQ302105）
T
YM27-005（AB297806）
T
99
99
YM21-132（AB297805）
T
HOact23（AB277853）
99
sp. MN1-1006（AB373024）
92
T
100
YM29-052（AB353310）
100
Marine clone SJC3.21（DQ071119）
Polychaete clone
A 54（AJ441243）
92
T
94
YM20-087（AB331886）
YM21-151
（AB331887）
100
T
MN1-741（AB331888）
T
YM27-120（AB331889）
100
T
YM26-010（AB331890）
63
YM24-184
（AB331891）
99
99
77
YM20-122（AB331892）
100
Elbe river clone DEV005（AJ401105）
Crater lake clone CL500-85（AF316731）
99
T
A5J-41-2（AB331893）
100
A5J-40
（AB331894）
T
57
A4T-83（AB331895）
99
T
YM23-227（AB372852）
98
97
T
93
06SJR1-1（AB372853）
73
T
AK18-024（AB372854）
T
05IJR53-1（AB372855）
T
99
MN1-1037（AB372856）
100
MN1-1047
（AB372857）
T
ATCC BAA-835（AY271254）
100
Human colonic clone HuCa18（AJ408970）
T
99
FC4 ATCC 25309（U60015）
T
100
FC1 ATCC 27091（U60012）
100
T
FC3i ATCC 700200（U60014）
T
FC2 ATCC 700199（U60013）
100
T
DSM 4136（X90515）
100
’
Ellin428（AY388649）
100
Bacterium Ellin502
（AY960765）
88
Bacterium Ellin507
（AY960770）
Uncultured bacterium clone DA101（Y07576）
92
99
Candidatus
（AF217461）
Candidatus
（AF217460）
100
Candidatus
（AF217462）
100 Bacterium Ellin515（AY960778）
100
Bacterium Ellin518（AY960781）
Bacterium Ellin5102（AY234519）
92
Freshwateriake clone LD19（AF009974）
T
PB90-1 DSM 11246（AJ229235）
100
ACB90（AJ229246）
98 46
PB90-3（AJ229239）
84
sp. VeGlc2（X99390）
100
sp. VeCb1
（X99391）
98
Archaeological wood clone pACH90（AY297806）
59
sp. strain VeSm13（X99392）
100
Opitutaceae bacterium TAV1（AY587231）
Uncultured bacterium clone BCf3-05（AB062814）
96
82 Opitutaceae bacterium TAV3（AY587233）
82
Opitutaceae bacterium TAV4（AY587234）
100
Opitutaceae bacterium TAV2（AY587232）
T
BCRC 19135（AF075271）
T
91
YM31-114（AB372849）
T
100
YM26-026（AB292183）
T
YM31-066（AB372850）
93
100
YM31-067
（AB372851）
62
T
IMCC1545（DQ539046）
T
04OKA010-24（AB266750）
37 100
‘
’
SI-1234（AB073978）
T
54
N5FB36-5（AB297922）
T
02PA-Ca-133（AB286015）
T
YM14-201（AB286016）
100
H-MN48
66
67
MN1-156
100
T
H-MN57（AB286017）
sp. SA-9（AY695840）
99
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）
T
ATCC 11775（X80725）
99
The class
The order
The family Verrucomicrobiaceae
（Subdivision 1）
The class
‘
（Subdivision 2）
’
Subdivision 3
Subdivision 6
The order
The family
The class
（Subdivision 4）
The order
The family
Subdivision 5
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.
─ 63 ─
Phylogenetic studies on the bacterial phylum‘Verrucomicrobia ’
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 N1-741 T ; 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 sargassicola 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’
, we formally proposed
Verrucomicrobia phy. nov., nom. rev. (Yoon et al.,
2010a).
CONCLUSION
We attempted to elucidate the phylogenetic relationships of the thirty novel isolates using polyphasic taxonomic approaches including 16S rRNA and
gyrβ gene sequence analysis, together with molecular, physiological, biochemical and chemotaxonomic
analyses to characterize the novel strains. Based on
these data, it is proposed that these isolates represent seven novel genera and twenty-two new spec 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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