Cerasicoccus maritimus sp. nov., and Cerasicoccus frondicus sp. nov., isolated from seawater and marine leaf, and proposal of phylum Verrucomicrobia phyl. nov.

Page 1

Introduction

　The phylum ‘Verrucomicrobia’ (Hedlund et al., 1997;

Hugenholtz et al., 1998) is one of the primary lineages within the domain Bacteria. They represent a distinct lineage within the phylogenetic trees and contain a

J. Gen. Appl. Microbiol., 56, 213‒222 (2010)

Three Gram-negative, pale-pink-pigmented, spherical, chemoheterotrophic bacteria were iso-lated from seawater and a dystrophic leaf in the Republic of Palau. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the novel isolates YM31-114T, YM31-066T and YM31-067 shared approximately 97‒100% sequence similarity with members of the genus Cerasicoc-cus of the family Puniceicoccaceae within the phylum ‘Verrucomicrobia.’ The hybridization val-ues for DNA‒DNA relatedness between the novel isolates and Cerasicoccus arenae YM26-026T were less than 70%, which is accepted as a phylogenetic defi nition of a species. β-Lactam anti-biotic susceptibility test and amino acid analysis of cell-wall hydrolysates revealed that the nov-el isolates did not contain muramic acid or diaminopimelic acid in their cell walls, suggesting that these strains lack peptidoglycan. The DNA G+C contents of the three strains were 55‒56 mol%; MK-7 was the major menaquinone. The presence of C14:0 and C18:1ω9c as the major cellular fatty acids supported the identifi cation of the novel isolates as members of the genus Cerasicoccus. On the basis of polyphasic taxonomic evidence, it was concluded that these strains should be classifi ed as representing two novel, separate species in the genus Cerasicoc-cus within the phylum ‘Verrucomicrobia,’ for which the names Cerasicoccus maritimus sp. nov. (type strain YM31-114T=MBIC24844T) and Cerasicoccus frondis sp. nov. (type strain YM31-066T=MBIC24796T) are proposed. Proposal for designation of the Verrucomicrobia phyl. nov., nom. rev. is also presented.

Key Words—β-lactam antibiotics; Cerasicoccus; in situ cultivation; peptidoglycan-less bacteria; Ver-

rucomicrobia

Full Paper

Cerasicoccus maritimus sp. nov. and Cerasicoccus frondis sp. nov., two peptidoglycan-less marine verrucomicrobial species, and

description of Verrucomicrobia phyl. nov., nom. rev.

Jaewoo Yoon,1,†, * Yoshihide Matsuo,2,†† Satoru Matsuda,2 Hiroaki Kasai,2,††† and Akira Yokota1

1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113‒0032, Japan2 Marine Biotechnology Institute Co., Ltd., Kamaishi, Iwate 026‒0001, Japan

(Received November 16, 2009; Accepted January 4, 2010)

　* Address reprint requests to: Dr. Jaewoo Yoon, Department of Biotechnology, The University of Tokyo, 1‒1‒1 Yayoi, Bun-kyo-ku, Tokyo 113‒8657, Japan.　Tel: +81‒3‒5841‒5162　　Fax: +81‒3‒5841‒8033　E-mail: [email protected]　† Present address: Department of Biotechnology, The Univer-sity of Tokyo, 1‒1‒1 Yayoi, Bunkyo-ku, Tokyo 113‒8657, Ja-

pan.　†† Suntory Holdings Limited, R&D Planning Division, 1‒1‒1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618‒8503, Japan.　†††Marine Biosciences Kamaishi Research Laboratory, Ki-tasato University, 3‒75‒1 Heita, Kamaishi, Iwate 026‒0001, Ja-pan.

Page 2

214 Vol. 56YOON et al.

number of environmental species as well as a small number of cultured species. Based on verrucomicro-bial 16S rRNA gene sequences and other molecular phylogenetic studies, members of the phylum ‘Verru-

comicrobia’ have been detected in a very wide range of quite different habitats within the global ecosystem (Dedysh et al., 2006; Hugenholtz et al., 1998; Joseph et al., 2003; O’Farrell and Janssen, 1999; Rappé and Giovannoni, 2003). Recently, a phylogenetic analysis of environmental metagenomic libraries indicated that many rRNA genes from the bacterial artifi cial chromo-some (BAC) library had substantially been affi liated with the bacterial phylum of ‘Verrucomicrobia’; all of the sequences were affi liated with subdivisions that lack cultured representatives (Liles et al., 2003). At present, these lineages are informally classifi ed into six monophyletic subdivisions numbered 1 to 6 (Vandekerckhove et al., 2000) of which three of the subdivisions are recognized in the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001) as the families Verrucomicrobiaceae (subdivision 1), Opitutaceae (subdivision 4) and ‘Xi-

phinematobacteriaceae’ (subdivision 2). Since the six informal monophyletic subdivisions were fi rst pro-posed, the names of only a few genera belonging to subdivisions 1 and 4 such as Akkermansia, Alterococ-

cus, Opitutus, Prosthecobacter, Rubritalea and Verru-

comicrobium have been validly published (Chin et al., 2001; Derrien et al., 2004; Hedlund et al., 1996, 1997; Scheuermayer et al., 2006; Schlesner, 1987; Shieh and Jean, 1998). The class Opitutae, comprising two orders: Puniceicoccales containing the family Punicei-

coccaceae and Opitutales containing the family Opitu-

taceae, was formally proposed recently for the classifi -cation of subdivision 4 (Choo et al., 2007). Currently, the class Opitutae incorporates the genera Alterococ-

cus, Coraliomargarita, Cerasicoccus, ‘Fucophilus,’ Opitutus, Pelagicoccus and Puniceicoccus (Chin et al., 2001; Choo et al., 2007; Sakai et al., 2003; Shieh and Jean, 1998; Yoon et al., 2007a, b, c, d). In addition to the named microorganisms, three taxonomically uncharacterized isolates of ‘ultramicrobacteria’ from rice paddy fi eld soil with 16S rRNA gene sequences closely related to Opitutus terrae have also been de-scribed (Chin et al., 1999; Janssen et al., 1997). Altero-

coccus agarolyticus ADT3T, formerly misclassifi ed as a member of the class Gammaproteobacteria, has been proposed to be included in the family Opituta-

ceae. The name of a microorganism ‘Fucophilus fu-

coidanolyticus’ SI-1234, which is able to degrade fu-coidan and is isolated from sea cucumbers (Sticopus

japonicus), has not yet been validly published. In spite of their wide ecological distribution in nature, however, owing to the problem of uncultivability, the classifi ca-tion of the phylum ‘Verrucomicrobia’ is still ambiguous and informal. For this reason, for formal classifi cation of the phylum ‘Verrucomicrobia,’ it is recommended that many verrucomicrobial species that thrive in a wide range of terrestrial, aquatic and marine habitats should be isolated and cultivated. Moreover, the re-maining subdivisions must be proposed formally by the rank of class. Also, the phylum ‘Verrucomicrobia’ should be proposed as an offi cial name.　In this study, we attempted to elucidate the phyloge-netic relationships of three novel isolates YM31-114T, YM31-066T and YM31-067 using a polyphasic taxo-nomic approach, including 16S rRNA gene sequence analysis. Additionally, with the inclusion of these iso-lates, we present a description of the phylum Verruco-

microbia for offi cial classifi cation of ‘Verrucomicrobia.’ Based on these data, it is proposed that these isolates represent two novel species of the genus Cerasicoc-

cus within the phylum ‘Verrucomicrobia.’

Materials and Methods

　Isolation of bacterial strains and cultivation.　Strain YM31-114T was isolated by using an in situ cultivation technique (Yasumoto-Hirose et al., 2006) from an arti-fi cial polyurethane foam (PUF) block supplemented with 1/10 strength medium ‘P’ (Yoon et al., 2007b) with 0.5% gellan gum containing 0.1% (w/v) lignin and 0.1% (w/v) vanillin. The PUF block was placed in the sea (GPS location, 07° 19′ 32.3″ N, 134° 29′ 26.2″ E, 10 m depth; Republic of Palau) for 3 days during April 2007. After 3 days, pieces of the PUF blocks (0.5‒1 cm3) were homogenized with a glass rod in 5 ml of sterile seawater. A 50 μl sample of the homogenate was ap-plied to the surface of an agar isolation medium. Strain YM31-114T appeared after 30 days incubation at 25°C on the medium ‘P’. The bacteria were purifi ed on ma-rine broth 2216 (Difco) containing 1.5% agar by culti-vation for 7‒10 days. Strains YM31-066T and YM31-067 were isolated from a dystrophic leaf collected in a marine lake near the Ulong Channel (GPS location, 07° 16′ 36.4″ N, 134° 17′ 28.3″ E, 1 m depth; Republic of Palau) in April 2007. 1/2 strength R2A agar (Difco) containing 75% artifi cial seawater (Lyman and Flem-

Page 3

2010 215Two novel species of the genus Cerasicoccus

ing, 1940) was used for cultivation and maintenance of the novel isolates.　Morphological, physiological and biochemical test.

Cell morphology was observed using light microscopy (BX60; Olympus). The temperature and pH ranges for growth were determined by incubating the isolates on 1/2 R2A agar (Difco) with 75% artifi cial seawater. The NaCl concentration for growth was determined on 1/2 R2A agar containing 0‒10% (w/v) NaCl. Gram-staining was performed as described by Murray et al. (1994). Growth under anaerobic conditions was determined after incubation for 2 weeks in an AnaeroPack (Mitsu-bishi Gas Chemical Co.) on 1/2 R2A agar with 75% artifi cial seawater. Catalase activity was determined by bubble formation in a 3% H2O2 solution. Oxidase activ-ity was determined using cytochrome oxidase paper (Nissui Pharmaceutical Co.). API 20E, API 50CH and API ZYM strips (bioMérieux) were used to determine the physiological and biochemical characteristics. All suspension media for the API test strips were supple-mented with 0.85% (w/v) NaCl solution (fi nal concen-tration). API 20E, API 50CH and API ZYM test strips were read after 72 h incubation at 30°C and 4 h incuba-tion at 37°C.　16S rRNA gene sequencing, phylogenetic analysis and

DNA‒DNA hybridization.　An approximately 1,500 bp fragment of the 16S rRNA gene was amplifi ed from the extracted DNA by using bacterial universal primers specifi c to the 16S rRNA gene: 27F and 1492R (Esch-

erichia coli numbering system; Weisburg et al., 1991). To ascertain the phylogenetic position of the novel iso-lates, the 16S rRNA gene sequences of strains YM31-114T, YM31-066T and YM31-067 were compared with the sequences obtained from GenBank (National Cen-ter for Biotechnology Information, http://www.ncbi.nlm.nih.gov). Multiple alignments of the sequences were performed using CLUSTAL_X (version 1.83) (Thompson et al., 1997). Alignment gaps and ambigu-ous bases were not taken into consideration when the 1,164 bases of the 16S rRNA gene nucleotides were compared. Aligned sequences were analyzed by us-ing MEGA3.1 software (Kumar et al., 2004). The evolu-tionary distances [distance options according to the Kimura two-parameter model (Kimura, 1983)] and clustering with the neighbor-joining (Saitou and Nei, 1987) and maximum-parsimony (Fitch, 1971) methods were determined by using bootstrap values based on 1,000 replications (Felsenstein, 1985). The similarity values were calculated using the same software.

DNA‒DNA hybridizations were carried out with photo-biotin-labeled probes in microplate wells as described by Ezaki et al. (1989). The hybridization temperature was set at 50°C. Hybridization was performed using fi ve replications for each. Of the values obtained, the highest and lowest for each sample was excluded and the means of the remaining three values are quoted as DNA‒DNA relatedness values.　Chemotaxonomic investigation.　Determination of the respiratory quinone system and cellular fatty acid composition were carried out as described previously (Katsuta et al., 2005). DNA was prepared according to the method of Marmur (1961) from cells grown on 1/2 strength R2A agar with 75% artifi cial seawater and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989). Cell walls were prepared by the methods described by Schleifer and Kandler (1972), and amino acids in an acid hydrol-ysate of the cell walls were identifi ed using TLC (Harp-er and Davis, 1979) and HPLC, as their phenylthiocar-bamoyl derivatives, with a model LC-10AD HPLC apparatus (Shimadzu) equipped with a Wakopak WS-PTC column (Wako Pure Chemical Industries) (Yokota et al., 1993).　Antibiotic susceptibility test.　The β-lactam antibiot-ic susceptibility test against the novel isolates was checked on 1/2 strength R2A agar with 75% artifi cial seawater, using 8 mm paper disc (Advantec) at the fol-lowing antibiotic concentrations: ampicillin (1, 10, 100, 500 and 1,000 μg ml-1), penicillin G (1, 10, 100, 500 and 1,000 μg ml-1), carbenicillin (1, 10, 100 and 500 μg ml-1), oxacillin (1, 10, 100 and 500 μg ml-1) and cephalothin (1, 10, 100 and 500 μg ml-1).　Nucleotide sequence accession numbers.　The GenBank/EMBL/DDBJ accession number for 16S rRNA gene sequences of strains YM31-114T, YM31-066T and YM31-067 are AB372849, AB372850 and AB372851, respectively.

Results and Discussion

Molecular phylogenetic analysis

　Comparative analysis of the 16S rRNA gene se-quences revealed that strains YM31-114T, YM31-066T and YM31-067 were phylogenetically affi liated with the genus Cerasicoccus with bootstrap values of 100% from both the neighbor-joining method and the maxi-mum-parsimony analysis (Fig. 1). Analysis of the 16S rRNA gene sequences also showed that the sequence

Page 4

216 Vol. 56YOON et al.

of strain YM31-114T had the highest similarity (98.7%) to that of strains YM31-066T and YM31-067, followed by the marine bacteria Cerasicoccus arenae YM26-026T (98.3%). Furthermore, the 16S rRNA gene se-quence similarities between strains YM31-066T and YM31-067 was 100%. These strains possessed a 97.9% sequence similarity with Cerasicoccus arenae YM26-026T. All other cultivated species of the phylum ‘Verrucomicrobia’ with validly published names were more distantly related, possessing 16S rRNA sequence similarity levels of 90% or less.　DNA‒DNA hybridization values between strain YM31-114T and YM31-066T and Cerasicoccus arenae YM26-026T were on average 17.6% and 15.7%. More-over, DNA‒DNA relatedness values between strain YM31-066T and Cerasicoccus arenae YM26-026T were 18.1%. These results strongly suggest that the strains YM31-114T and YM31-066T should be classifi ed as two separate species (Wayne et al., 1987).

Morphological, physiological and biochemical analy-

sis

　Cells of the strains YM31-114T, YM31-066T and YM31-067 on 1/2 strength R2A agar with 75% artifi cial seawater were spherical and mostly 0.8‒1.5 μm in di-ameter. The cells did not bear fl agella or appendages. No motility was seen by light microscopy. Cells divided by means of binary fi ssion.　The strains YM31-114T, YM31-066T and YM31-067 also showed distinct phenotypic features that discrimi-nated them from the cultivated members of the class Opitutae given in Table 1.

Chemotaxonomic and cell wall peptidoglycan analysis

　As shown in Table 2, the predominant cellular fatty acids of novel strains were C14:0 (45.8‒52.8%) and C18:1ω9c (29.0‒36.5%), which are similar to other members of the genus Cerasicoccus. In addition, on the basis of their fatty acid composition, these strains are differentiated from Coraliomargarita akajimensis

Fig. 1.　Neighbor-joining phylogenetic tree based on 16S rRNA gene sequence analysis show-ing the positions of strains YM31-114T, YM31-066T and YM31-067 in relation to representative 16S rRNA gene sequences that include the currently known cultivated phylogenetic diversity within the class Opitutae of the phylum ‘Verrucomicrobia.’　Bootstrap values from both neighbor-joining (above nodes) and maximum-parsimony (below nodes) are shown. Sequences determined in this study are shown in bold. The sequence of Es-

cherichia coli ATCC 11775T was used as an outgroup. Bar, 2% sequence divergence.

Page 5

2010 217Two novel species of the genus CerasicoccusTa

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218 Vol. 56YOON et al.Ta

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-N

D

　Ta

xa:

1, s

trai

n Y

M31

-114

T ; 2,

str

ain

YM

31-0

66T ;

3, s

trai

n Y

M31

-067

; 4,

Ce

rasic

oc

cu

s a

ren

ae

YM

26-0

26T (

data

fro

m Y

oon

et a

l., 2

007c

); 5

, C

ora

liom

arg

ari

ta a

kajim

en

sis

04

OK

A01

0-24

T (Yo

on e

t al.,

200

7a);

6, P

ela

gic

oc

cu

s m

ob

ilis 0

2PA

-Ca-

133T (

Yoon

et a

l., 2

007b

); 7

, Pe

lag

ico

cc

us a

lbu

s Y

M14

-201

T (Yo

on e

t al.,

200

7b);

8, P

ela

gic

oc

cu

s lito

ra-

lis H

-MN

57T (

Yoon

et a

l., 2

007b

); 9

, Pe

lag

ico

cc

us c

roc

eu

s N

5FB

36-5

T (Y

oon

et a

l., 2

007d

); 1

0, P

un

ice

ico

cc

us v

erm

ico

la IM

CC

1545

T (C

hoo

et a

l., 2

007)

.　

Dat

a ar

e ex

pres

sed

as p

erce

ntag

es o

f tot

al fa

tty a

cids

. Fat

ty a

cids

rep

rese

ntin

g le

ss th

an 1

% a

re n

ot s

how

n. -

, Not

det

ecte

d; N

D, n

ot d

escr

ibed

; tr,

trac

e.

Tabl

e 3.　

Am

ino

acid

ana

lysi

s of

cel

l-wal

l hyd

roly

sate

s an

d β-

lact

am a

ntib

iotic

sus

cept

ibili

ty te

st o

f the

cla

ss O

pitu

tae

with

in th

e ph

ylum

‘Ve

rru

co

mic

rob

ia.’

Cha

ract

eris

tic1

23

45

67

89

1011¶

1213

Cel

l wal

l com

pone

nt o

f:　

Mur

amic

aci

d-

--

--

--

--

--

++

　m

eso

-Dia

min

opim

elic

aci

d-

--

--

--

--

--

++

Ant

ibio

tic r

esis

tant

(μg

ml-

1 ) to

:　

Am

pici

llin

(1‒1

,000

)+

++

++

++

++

+N

D-

-　

Pen

icill

in G

(1‒

1,00

0)+

++

++

++

++

+N

D-

-　

Car

beni

cilli

n (1‒5

00)

++

++

++

++

++

ND

--

　O

xaci

llin

(1‒5

00)

++

++

++

++

++

ND

--

　C

epha

loth

in (

1‒50

0)+

++

++

++

++

+N

D-

-

　¶ A

naer

obic

exp

erim

enta

l cul

tivat

ion

was

per

form

ed a

ccor

ding

to th

e m

etho

d of

Chi

n et

al.

(199

8).

　Ta

xa: 1

, str

ain

YM

31-1

14T ; 2

, str

ain

YM

31-0

66T ; 3

, Ce

rasic

oc

cu

s a

ren

ae

YM

26-0

26T (

data

from

Yoo

n et

al.,

200

7c);

4, C

ora

liom

arg

ari

ta a

kajim

en

sis

04O

KA

010-

24T (

Yoon

et

al.,

2007

a); 5

, Pe

lag

ico

cc

us m

ob

ilis 0

2PA

-Ca-

133T (

Yoon

et a

l., 2

007b

); 6

, Pe

lag

ico

cc

us a

lbu

s Y

M14

-201

T (Yo

on e

t al.,

200

7b);

7, P

ela

gic

oc

cu

s lito

ralis

H-M

N57

T (Yo

on e

t al.,

20

07b)

; 8, P

ela

gic

oc

cu

s c

roc

eu

s N

5FB

36-5

T (Yoo

n et

al.,

200

7d);

9, P

un

ice

ico

cc

us v

erm

ico

la IM

CC

1545

T (thi

s st

udy)

; 10,

Alte

roc

oc

cu

s a

garo

lytic

us B

CR

C 1

9135

T (thi

s st

udy)

; 11

, Op

itu

tus te

rrae

DS

M 1

1246

T (th

is s

tudy

) ¶ ;

12,

Ro

se

ibac

illu

s p

on

ti Y

M27

-120

T (Yo

on e

t al.,

200

8); 1

3, R

ose

ibac

illu

s p

ers

icic

us Y

M20

-122

(Yo

on e

t al.,

200

8).

　+

, Det

ecte

d or

res

ista

nt; -

, not

det

ecte

d or

sus

cept

ible

; ND

, no

data

.

Page 7

2010 219Two novel species of the genus Cerasicoccus

04OKA010-24T, Pelagicoccus spp. and Puniceicoc-

cus vermicola IMCC1545T, their phylogenetically neighboring taxa, indicating that strains YM31-114T, YM31-066T and YM31-067 probably represent two in-dependent species of the genus Cerasicoccus of the family Puniceicoccaceae within the phylum ‘Verruco-

microbia.’　When the novel isolates were grown in the presence of increasing concentrations (1‒500 or 1‒1,000 μg ml-1) of the β-lactam antibiotics, ampicillin, penicillin G, carbenicillin, oxacillin and cephalothin, they showed a remarkable resistance (Table 3).　The cell walls of all the novel isolates were prepared by disruption of cells, followed by heating with 3% SDS, washing and centrifugation. Amino acid analysis of the cell-wall hydrolysates indicated the absence of muramic acid and diaminopimelic acid in the cell wall, which suggests that the strains do not contain an ordi-nary Gram-negative type of peptidoglycan in their cell walls (Table 3).

In conclusion

　Based on the results of the molecular phylogenetic analysis and their biochemical and physiological prop-erties, the three novel strains YM31-114T, YM31-066T and YM31-067 should be classifi ed as representing two independent species of the genus Cerasicoccus. We propose the names Cerasicoccus maritimus sp. nov. (type strain YM31-114T) and Cerasicoccus frondis sp. nov. (type strain YM31-066T).

Description of Verrucomicrobia phyl. nov., nom. rev. (Hedlund et al., 1997, emend. Garrity and Holt) Yoon et al., 2010　Verrucomicrobia (Ver.ru.co.mi.cro’bi.a N.L. fem. pl. n. Verrucomicrobiales type order of the phylum, drop-ping ending to denote a phylum; N.L. fem. pl. n. Ver-

rucomicrobia phylum of Verrucomicrobiales).　Equivalent to the phylum ‘Verrucomicrobia’ and de-fi ned by phylogenetic analyses based on 16S rRNA gene sequences obtained from several cultivated members and a wide range of uncultivated bacteria retrieved mainly from soil, aquatic and marine habi-tats. Gram-negative. The phylum comprises the class Verrucomicrobiae and Opitutae and four subdivisions. The type order is order Verrucomicrobiales (Ward-Rainey et al., 1995).

Description of Cerasicoccus maritimus sp. nov.　Cerasicoccus maritimus (ma.ri’ti.mus. L. masc. adj. maritimus pertaining to the sea).　Cells are cocci, 0.8‒1.5 μm in diameter. Neither cel-lular gliding movement nor swarming growth is ob-served. Colonies grown on 1/2 strength R2A agar with 75% artifi cial seawater medium are circular, convex and pale pink in color. The temperature range for growth is 10‒37°C, optimally at 25‒30°C, but no growth occurs at 4 or 45°C. The pH range for growth is 6‒9. Seawater is not required for growth. NaCl is not re-quired for growth, but can be tolerated up to 5% (w/v). Growth occurs in the presence of ampicillin (1‒1,000 μg ml-1) and penicillin G (1‒1,000 μg ml-1), car-benicillin (1‒500 μg ml-1), oxacillin (1‒500 μg ml-1) and cephalothin (1‒500 μg ml-1). Esculin and gela- tin are hydrolyzed, but starch, agar, DNA and urea are not hydrolyzed. The reactions for tryptophan deami-nase are positive, but acetoin, citrate utilization, argi-nine dihydrolase, lysine decarboxylase, ornithine de-carboxylase, hydrogen sulfi de and indole production are negative. Acid is produced from esculin ferric cit-rate, lactose, gentiobiose, and L-arabinose, but not from D-lyxose, galactose, glucose, fructose, mannose, methyl-α-D-glucopyranoside, D-turanose, 5-keto-glu-conate, trehalose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, erythritol, mannitol, sorbitol, glyc-erol, D-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl-β-D-xylopyranoside, sorbose, rhamnose, dulci-tol, inositol, methyl-α-D-mannnopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, melibiose, sucrose, inulin, melezitose, raffi -nose, starch, glycogen, xylitol, gluconate or 2-keto-gluconate. Alkaline phosphatase, chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase are positive, but β-galactosidase, α-galactosidase, α-glucosidase, leucine arylamidase, valine arylami-dase, trypsine, esterase (C4), esterase lipase (C8), li-pase (C4), cystine arylamidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-man-nosidase and α-fucosidase are negative. The usual components of bacterial cell walls such as muramic acid and diaminopimelic acid are not detected. Major fatty acid components (＞1.0%) include C14:0 (45.8%), C15:0 (2.0%), C14:0 2-OH (1.9%), C16:1ω7c (2.6%), C16:0 (6.2%), C18:1ω9c (36.5%) and C18:0 (2.1%). The DNA G+C content of the type strain is 55.7 mol%. The type strain is YM31-114T (=MBIC24844T), which was iso-lated from seawater by using an in situ cultivation tech-

Page 8

220 Vol. 56YOON et al.

nique.

Description of Cerasicoccus frondis sp. nov.　Cerasicoccus frondis (fron’dis. L. gen. n. frondis of a leaf).　Cells are cocci, 0.8‒1.5 μm in diameter. Neither cel-lular gliding movement nor swarming growth is ob-served. Colonies grown on 1/2 strength R2A agar with 75% artifi cial seawater medium are circular, convex and pale pink in color. The temperature range for growth is 10‒37°C, optimally at 30‒37°C, but no growth occurs at 4 or 45°C. The pH range for growth is 7‒9. Seawater is not required for growth. NaCl is not re-quired for growth, but can be tolerated up to 6% (w/v). Growth occurs in the presence of ampicillin (1‒1,000 μg ml-1) and penicillin G (1‒1,000 μg ml-1), car-benicillin (1‒500 μg ml-1), oxacillin (1‒500 μg ml-1) and cephalothin (1‒500 μg ml-1). Esculin and gela- tin are hydrolyzed but, starch, agar, DNA and urea are not hydrolyzed. The reactions for ONPG and trypto-phan deaminase are positive, but acetoin, citrate utili-zation, arginine dihydrolase, lysine decarboxylase, or-nithine decarboxylase, hydrogen sulfi de and indole production are negative. Acid is produced from L-ara-binose, D-xylose, galactose, glucose, fructose, and mannose, but not from methyl-α-D-glucopyranoside, esculin ferric citrate, lactose, gentiobiose, D-turanose or 5-keto-gluconate, trehalose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, erythritol, mannitol, sor-bitol, glycerol, D-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl-β-D-xylopyranoside, sorbose, rham-nose, dulcitol, inositol, methyl-α-D-mannopyranoside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, cel-lobiose, maltose, melibiose, sucrose, inulin, melezito-se, raffi nose, starch, glycogen, xylitol, gluconate or 2-keto-gluconate. Alkaline phosphatase, acid phos-phatase, naphthol-AS-BI-phosphohydrolase, β-gala-ctosidase and β-glucosidase are positive, but α-gala-ctosidase, α-glucosidase, leucine arylamidase, valine arylamidase, trypsine, esterase (C4), esterase lipase (C8), lipase (C4), cystine arylamidase, chymotrypsin, β-glucuronidase, N-acetyl-β-glu cosaminidase, α-man-nosidase and α-fucosidase are negative. The usual components of bacterial cell walls such as muramic acid and diaminopimelic acid are not detected. Major fatty acid components (＞1.0%) include iso-C14:0 (3.5%), C14:0 (38.7%), C14:0 2-OH (4.4%), C16:0 (2.3%), C16:0 3-OH (1.3%), C18:1ω9c (43.3%), C18:0 (1.5%) and C20:0 (1.5%). The G+C content of the DNA of the type

strain is 56.8 mol%. The type strain is YM31-066T (= MBIC24796T), which was isolated from a dystrophic leaf in a marine lake.

Acknowledgments

　We would like to thank Atsuko Katsuta, Ayako Matsuzaki, To-momi Haga, Yukiko Itazawa, and Jeung-yil Park for their techni-cal assistance. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).

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