Chryseobacterium Oleae Sp. Nov., an Efficient Plant

Systematic and Applied Microbiology 37 (2014) 342–350

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Systematic and Applied Microbiology

j ournal homepage: www.elsevier.de/syapm

Short communication

Chryseobacterium oleae sp. nov., an efﬁcient plant growth promoting

bacterium in the rooting induction of olive tree (Olea europaea L.)

cuttings and emended descriptions of the genus Chryseobacterium,

C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum,

C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense

a,b,∗∗ a c

Maria del Carmen Montero-Calasanz , Markus Göker , Manfred Rohde ,

a a d e

Cathrin Spröer , Peter Schumann , Hans-Jürgen Busse , Michael Schmid ,

a a,∗ b

Hans-Peter Klenk , Brian J. Tindall , Maria Camacho

Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany

IFAPA-Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres-Tomejil, Ctra, Sevilla-Cazalla de la Sierra, Km 12.2, Alcalá del Río,

41200 Sevilla, Spain

HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany

Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria

Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: A novel non-motile, Gram-staining-negative, yellow-pigmented bacterium, designated CT348 , isolated

Received 5 November 2013

from the ectorhizosphere of an organic olive tree in Spain and characterised as an efﬁcient plant growth

Received in revised form 21 February 2014

promoting bacterium, was investigated to determine its taxonomic status. The isolate grew best in a

Accepted 22 April 2014 ◦

temperature range of 5–35 C, at pH 5.0–8.0 and with 0–1% (w/v) NaCl. Chemotaxonomic and molecular

characteristics of the isolate matched those described for members of the genus Chryseobacterium. The

Keywords:

DNA G + C content of the novel strain was 38.2 mol%. The strain contained a polyamine pattern with

Bacteriodetes

sym-homospermidine as the major compound and produced ﬂexirubin-type pigments. MK-6 was the

Flavobacteriaceae

␻

Taxonomy dominant menaquinone and the major cellular fatty acids were iso-C15:0, C17:1 9c, iso-C17:0 3-OH and

PGPB iso-C15:0 2-OH. The main polar lipids were phosphatidylethanolamine and several unidentiﬁed lipids and

Indole-3-acetic acid aminolipids. The 16S rRNA gene showed 92.2–97.8% sequence identity with the members of the genus

Chyseobacterium. Based on the phenotypic traits and DNA–DNA hybridizations with the type strains of

the most closely related species, the isolate is shown to represent a novel species, Chyseobacterium oleae,

type strain CT348 (=DSM 25575 =CCUG 63020). Emended descriptions of the genus Chryseobacterium

and C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum, C. shigense, C. taiwanense, C.

ureilyticum and C. vrystaatense are also proposed.

The genus Chryseobacterium [58] whose type species is members of the genus Chryseobacterium based on the interpre-

Chryseobacterium gleum [21,58] is included within the family tation of the list of all validly published names within this genus

Flavobacteriaceae (emended by Bernardet et al. [4]), the main according to the “List of Prokaryotic Names with Standing in

bacterial lineage in the phylum Bacteroidetes. At the time of writing Nomenclature” (http://www.bacterio.net/). Members of the genus

76 species with validly published names were considered to be Chryseobacterium can be found in a wide variety of environments,

including freshwater [50], soil [32], rhizosphere [9], phyllosphere

[3], sludge [43], ﬁsh [68–70], midgut of insects [26], raw dairy

∗ products [19], raw chicken [11], faeces of millipede [27], industrial

Corresponding author. Tel.: +49 0531 26 16 224; fax: +49 0531 26 16 418.

∗∗ plants [20] and clinical samples [67].

Corresponding author. Tel.: +49 0531 26 16 210; fax: +49 0531 26 16 418.

Based on previous studies, it is known that members of the

E-mail addresses: [email protected] (M.d.C. Montero-Calasanz),

[email protected] (B.J. Tindall). genus Chryseobacterium are considered to be an important bacterial

http://dx.doi.org/10.1016/j.syapm.2014.04.004

M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350 343

group associated with plants [1,7,31] and currently there is strong were performed as described in Tindall et al. [55]. The utilisation

evidence to show that strains of plant-associated Chryseobacterium of carbon compounds and acid production were determined using

species exhibit plant-growth promoting activities [10,38]. API 20NE and API 20E strips (bioMérieux) according to the manu-

◦

During the characterisation of organisms isolated from the rhi- factures instructions, incubated at 28 C as well as using GEN III

zosphere of an organically farmed olive grove (Olea europaea L. Microplates incubated in an Omnilog device (BIOLOG Inc., Hay-

◦

cv. arbequina) in Mairena del Aljarafe, Seville, Spain (37 20 18.97 ward, CA, USA). The GEN III Microplates were inoculated with a

◦

N, 6 5 8.61 W), in 2006, for application in alternative systems to cell suspension made in “gelling” inoculating ﬂuid (IF) A at a cell

produce organically grown olive plants, strain CT348 was recov- density of 90% transmittance, yielding a running time of 4 days

◦

ered. This study based on a polyphasic approach in agreement with in Phenotype Microarray mode at 28 C. The exported data were

the recommended minimal standards for describing new taxa of further analysed with the opm package for R [56,57] v.0.9.23.,

the family Flavobacteriaceae [4] refers the taxonomic position of using its functionality for merging subsequent measurements of

a plant growth promoting bacterial (PGPB) strain representing a the same plate, statistically estimating parameters from the res-

novel species within the genus Chryseobacterium isolated from the piration curves such as the maximum height, and automatically

ectorhizosphere of an organically farmed olive tree. “discretizing” these values into negative and positive reactions.

Ectorhizospheric soil samples were suspended and Strain CT348 in comparison with the reference strains Chryseobac-

homogenised in sterile mineral buffer [59]. Serial dilutions terium daecheongense DSM 15235 , Chryseobacterium deﬂuvii DSM

TM T T

were inoculated on Plate Count Agar (PCA; Difco ) diluted at 50% 14219 , Chryseobacterium gambrini DSM 18014 , Chryseobacterium

T T

supplemented with cycloheximide (100 mg/l), adjusted at pH 7.0, gleum DSM 16776 , Chryseobacterium gregarium DSM 19109 , Chry-

◦ T T

at 28 C for 5 days. Strain CT348 was ﬁrstly isolated based on seobacterium indologenes DSM 16777 , Chryseobacterium joostei

T T

colony morphology, puriﬁed by sub-culturing on Trypticase Soy DSM 16927 , Chryseobacterium jejuense DSM 19299 , Chryseobac-

TM T T

Agar (TSA; Difco ) diluted at 10% and then selected by its ability terium luteum DSM 18605 , Chryseobacterium shigense DSM 17126 ,

to produce indole-3-acetic acid (IAA) and siderophores, desirable Chryseobacterium taiwanense JCM 21767 , Chryseobacterium ure-

T T

PGPR features, and to promote rooting in model plants and olive ilyticum DSM 18017 Chryseobacterium vrystaatense DSM 25206

cuttings [37]. The culture was suspended in 0.5% peptone and 15% and Chryseobacterium wanjuense DSM 17724 was just studied in

◦

(w/v) glycerol for storage at −80 C. the GEN III Microplates, in two independent determinations. Reac-

The colony morphology and pigmentation of strain CT348 tions that gave contradictory results between the two repetitions

were observed on TSB agar after 24 and 72 h under a binocular were regarded as ambiguous.

microscope according to Pelczar [42]. Exponentially growing bac- The extraction and analysis of cellular fatty acids was carried

◦

terial cultures were observed with an optical microscope (Zeiss out from biomass grown on TSA plates held at 28 C for 24 h and

AxioScope A1) with a 100-fold magniﬁcation and phase-contrast harvested always from the same sector (the last quadrant streak).

illumination. Micrographs of bacterial cells grown on Trypticase Analysis was conducted using the Microbial Identiﬁcation System

Soy Broth (TSB) for 48 h were taken with a ﬁeld-emission scanning (MIDI) Sherlock Version 6.1 (results evaluated against the TSBA40

electron microscope (FE-SEM Merlin, Zeiss, Germany). Gram stain- peak naming table database) as described by Sasser [46]. The fatty

ing was performed according to Halebian et al. [15] and checked by acid extracts obtained using the standard MIDI protocol were sub-

the KOH test [14]. Anaerobic respiration was investigated by incu- jected to further analysis by GC–MS. GC–MS experiments were

TM ◦

bation in an anaerobic pouch (Merck ) for 7 days at 28 C on 1% carried out using a Varian CP-3800 gas chromatograph, ﬁtted with

casitone, 0.3% yeast extract broth and 0.2% nitrate and 0.2% nitrite a Varian CP-8400 autosampler, coupled to a Varian 320-MS mass

as terminal electron acceptors [65]. Activity of oxidase was ana- spectrometer. The gas chromatograph was ﬁtted with a 30 m Varian

lysed using ﬁlter-paper disks (Sartorius grade 388) impregnated VF-5 ms capillary column, internal diameter 0.25 mm, ﬁlm thick-

◦

with 1% solution of N,N,N ,N -tetramethyl-p-phenylenediamine ness 0.25 ␮m. The run conditions were injector temperature 250 C,

◦ ◦ ◦

(Sigma–Aldrich); a positive test was indicated by the immediate transfer line 260 C, ion source 250 C, column temperature 130 C

◦ ◦

development of a blue-purple colour after applying biomass on the to 250 C at a rate of 4 C per minute, carrier gas helium at a con-

filter paper. Catalase activity was tested by the observation of bub- stant flow rate of 1.3 ml/min. The final column temperature for

bles following the addition of drops of 3% H2O2. The KOH test to experiments on the trimethylsilyl (TMS) derivatives was increased

T ◦

assess whether the pigments produced by strain CT348 were of to 300 C. Spectra were recorded in the EI positive mode at 70 eV

the ﬂexirubin type was performed according Bernardet et al. [4]. over the m/z range 50–500. Trimethylsilyl derivatives were syn-

Growth rates were determined on plates of TSB agar for tempera- thesised by mixing 100 ␮l of the fatty acid methyl esters with 50 ␮l

◦ ◦

tures from 0 to 45 C in steps of 5 C and for pH values 4.0–12.5 (in of N, O-bis(trimethylsilyl)acetamide (BSA) and 50 ␮l pyridine, fol-

◦

steps of 0.5 pH units) using the buffer system described by Xu et al. lowed by heating at 50 C for 30 min. Polar lipids were extracted and

[64]. Degradation of speciﬁc substrates was examined using agar separated by two-dimensional thin-layer chromatography (TLC)

plates with various basal media, where results were considered as according to Bligh and Dyer [6] and Tindall et al. [55] and iden-

positive with the appearance of clear zones around the colonies: tiﬁed by Minnikin et al. [35] as modiﬁed by Kroppenstedt and

casein degradation was tested on medium containing skimmed Goodfellow [29]. Respiratory lipoquinones were extracted from

milk (5%) and NaCl (0.5%) solidiﬁed with 1% agarose; tyrosine degra- freeze-dried cell material with methanol:hexane as described by

dation was investigated as previously described [13] on plates Tindall [53,54], separated into their functional classes by TLC and

containing peptone (0.5%), beef extract (0.3%), l-tyrosine (0.5%) and analysed by reverse phase HPLC [53,54]. Analysis of cellular fatty

agarose (1.5%); the decomposition of xanthine and hypoxanthine acids, polar lipids and respiratory lipoquinones were carried out

was investigated by the same test, replacing l-tyrosine by hypo- using the reference strains listed above in parallel experiments.

xanthine or xanthine (0.4%), respectively; starch degradation was Polyamine were extracted as reported by Busse and Auling [8] and

tested on plates containing nutrient broth (Difco ) (0.8%), starch analysed according to Stolz et al. [49] using biomass cultivated on

(1%) and agarose (1.5%), developing these plates by ﬂooding with PYE broth (0.3% peptone from casein, 0.3% yeast extract at pH 7.2,

◦

iodine solution (1%) after incubation for 5 days. Enzyme activities 28 C and 180 rpm) and harvested at 70% of the maximum optical

of strain CT348 were tested using API ZYM galleries according to density.

the instructions of the manufacturer (bioMérieux). Other biochem- The G + C content of the chromosomal DNA was determined by

ical tests, such as cellulase activity (method 15.2.18), methyl red HPLC according to Mesbah et al. [34]. Chromosomal DNA was iso-

(method 15.2.52) and Voges–Proskauer (method 15.2.82) reactions lated with Wizard Genomic DNA Puriﬁcation Kit (Promega). The

344 M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350

16S rRNA coding gene was ampliﬁed by PCR using universal primers and C. daecheongense should be emended, as well as those of

◦

27f and 1492r [30] at an annealing temperature of 55.5 C. Ampli- the species C. joostei, C. luteum, C. shigense and C. vrystaatense,

ﬁed DNA fragments were cloned using the TOPO TA cloning kit since these results were missing from the original descriptions.

2.1 (Invitrogen). The sequencing of puriﬁed plasmid DNA was per- In the emended description of the genus Chryseobacterium [63]

formed by BigDye Terminator v.3.1 cycle sequencing kit (Applied reported only the presence of phosphatidylethanolamine, as the

Biosystems) as directed by the manufacturer’s protocol and ABI major phospholipid, nevertheless our data demonstrate total lipids

PRISM 3730 DNA analyser (Applied Biosystems). Sequence data proﬁles of Chryseobacterium species that contain three additional

were edited and assembled manually using BIOEDIT (Sequence unknown lipids (L1, L6 and L8) and two additional unidentiﬁed

Alignment Editor) v.7.0.5 [16]. Phylogenetic analysis was carried aminolipids (AL1 and AL2) (Fig. 1). Thus, the description of the

out according to Montero-Calasanz et al. [36]. Pairwise similarities genus Chyseobacterium should be emended to include the presence

were calculated from exact pairwise sequence alignments using the of these compounds, even if their structures remain to be deter-

Smith–Waterman algorithm as implemented in the EMBOSS suite mined. The absence of additional phospholipids lipids indicates

[44]. DNA–DNA hybridisation tests were performed in duplicate as phosphosphingolipids were absent, in line with the original genus

described by De Ley et al. [12] with the modiﬁcations suggested by description [58]. Bajerski et al. [2] have suggested that an ornithine

Huss et al. [23] using a Cary 100 Bio UV/VIS. lipid may be present in Chryseobacterium frigidisoli, however this

Cells of strain CT348 were Gram-staining-negative, non- is contradicted by the absence of 3-OH fatty acids (a pre-requisite

motile rods, frequently paired rods, with cell dimensions of for ornithine based lipids) in the fatty acid spectrum in that work,

0.4–0.5 × 1.4–2.8 ␮m (Supplementary Fig. S1), non-spore-forming which also deviates signiﬁcantly from the fatty acid pattern of all

and aerobic. Nitrate and nitrite were not used as terminal elec- known members of the genus Chryseobacterium.

tron acceptors. The colonies were convex, circular and translucent The major fatty acids were the saturated branched-chain

with a shiny, smooth and mucoid surface and entire edges. A non- acid iso-C15:0 (44.4%), the monounsaturated iso-C17:1␻9c (20.4%,

diffusible yellow-orange pigment that belongs to the ﬂexirubin although the annotation may be incorrect, see below) and the

T ◦

type was produced. Strain CT348 grew best at 5–35 C; no growth hydroxylated iso-C17:0 3-OH (16.4%) complemented by a fatty acid

◦ ◦

was observed below 5 C and above 35 C. Growth was observed in with an ECL that indicates the presence of iso-C15:0 2-OH (9.0%)

the presence of 0–1% NaCl but not 4% NaCl and at pH 5.0–8.0. The despite the fact that it is annotated as part of summed feature 3

full phenotype microarrays obtained using the OmniLog device in (iso-C15:0 2-OH/C16:1␻7c in the TSBA40 peak naming tables) and

comparison to the type strains of other Chryseobacterium species is in agreement with the fatty acid patterns obtained for the other

showed the distinction of strain CT348 from related species (Sup- Chryseobacterium species investigated in this study (Table 2). The

plementary Fig. S2); a summary of some differential phenotypic presence of a methyl ester of a 2-OH pentadecanoic fatty acid

characteristics is presented in Table 1. was conﬁrmed by mass spectrometry of the native compound and

Supplementary ﬁgures related to this article can be found, in the the trimethyl silyl derivative. The native fatty acid methyl ester

+ +

online version, at http://dx.doi.org/10.1016/j.syapm.2014.04.004. gave M at m/z 272 and (M-59) at m/z 213. The trimethyl silyl

Strain CT348 contained primarily menaquinone MK-6 (89.9%), derivative shifted to longer retention times and gave characteristic

+ +

a feature shared by all members of the family Flavobacteriaceae fragments at (M-15) m/z 329 and (M-59) m/z 285. No evidence

[5], but also MK-5 (10.1%) as well as C. daecheongense (6.8%), C. was found for the presence of any isomers the methyl esters of

deﬂuvii (4.9%), C. gambrini (2.8%), C. gleum (2.8%), C. gregarium hexadecenoic acid. It should be noted that earlier work on the fatty

(3.0%), C. indologenes (4.5%), C. joostei (10%), C. jejuense (9.5%), acids of members of the genus Chryseobacterium (also previously

C. luteum (3.7%), C. shigense (5.1%) and C. ureilyticum (9.6%), a referred to as CDC group IIb) unambiguously detected the pres-

component already detected in other Chryseobacterium species ence of iso-C15:0 2-OH by mass spectrometry [39,66] conﬁrmed

[38,51,68]. Polyamine analysis revealed as a major compound recently by Herzog et al. [20] whereas more recent reports of

sym-homospermidine (53.3 ␮mol), minor amounts of spermidine new species no longer record the presence of this fatty acid (see

(4.1 ␮mol) and spermine (3.2 ␮mol) and traces of putresceine for example [32,40,41,45,68–70]), instead reporting summed fea-

(0.2 ␮mol) and cadaverine (0.1 ␮mol), which is consistent with the ture 3 (=C16:1␻6c/C16:1␻7c) when using the TSBA6 peak naming

characteristic pattern for Chyseobacterium spp. [17,18,25,38]. The tables, highlighting a misleading annotation that regularly escapes

polar lipid proﬁle onsisted of the predominant compounds phos- attention and suggests that proper reference is not made to either

phatidylethanolamine (PE), two unknown lipids (L6 and L8) and an earlier or more recent relevant literature. The literature on mem-

unknown aminolipids AL1 with Rf values documented in Fig. 1a. bers of the genus Chryseobacterium uses the annotation summed

Furthermore, moderate amounts of four unknown lipids (L1, L5, feature 3 and summed feature 4 for a group of fatty acids labelled

L10 and L13) and two aminolipids AL2 and AL3 were detected. It as containing iso-C15:0 2-OH and different isomers of C16:1, some-

is also worth mentioning the presence of traces of an unknown times in the same table (see [5]), further adding to the confusion.

APL. This is in accordance with chromatographic proﬁles observed It should, however, be noted that the MIDI system is designed to

for reference strains in this study and those published by Kämpfer identify microorganisms based on their fatty acids patterns and

et al. [24,25], Herzog et al. [20] and Montero-Calasanz et al. [38], not necessarily to provide a comprehensive annotation of the fatty

although it should be noted that in the original descriptions of C. acids present. This also has implications when one refers back to

gambrini [20], L8 and AL4 were annoted as AL and L, respectively, the original genus description [58] and well as the elucidation of

and L1, L3–L5, L7, L9, L19, AL5–AL7 and APL were omitted (Fig. 1g). the underlying chemical structures from which they are derived.

Similarly, in the polar lipid proﬁles of C. ureilyticum [20] L1, L3, L4, As more genomes become available it should be possible to co-

L5, L7, L10 and AL6 were also not reported. On the other hand, Wu relate some of the critical genes involved in fatty acid synthesis that

et al. [63] recently showed polar lipid proﬁles for C. gambrini, C. modify precursor saturated fatty acids by the addition of points of

jejuense and C. taiwanense that appear to be inconsistent with those unsaturation (either during chain elongation or post-elongation via

reported here and in previous work. In addition, the paper describ- desaturases) or the addition of 2-hydroxy groups. Misleading fatty

ing C. daecheongense [28] did not provide a ﬁgure documenting acid annotations are clearly problematic. Similarly, some of the

the total polar lipids, the absence of which make it impossible more recent evaluations also no longer report the presence of only

to verify the consistency of the data included in the text with iso-C17:1␻9c, but now record it as part of summed feature 9. The

the data obtained. Based on the data presented here the species annotation of the iso-C17:1␻9c fatty acid differs in both earlier pub-

description of C. gambrini, C. ureilyticum, C. jejuense, C. taiwanense lications [39,62] as well as the work of Herzog et al. [20] from that

M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350 345

Fig. 1. Polar lipids proﬁle of Chryseobacterium oleae sp. nov. CT348 (a), C. daecheongense (b), C. gambrini (c), C. gleum (d), C. jejuense (e), C. joostei (f), C. luteum (g), C. shigense

(h), C. taiwanense (i), C. ureilyticum (j) and C. vrystaatense (k) after separation by two-dimensional TLC using the solvents chloroform:methanol:water (65:25:4; v:v:v) in the

ﬁrst dimension and chloroform:methanol:acetic acid:water (80:12:15:4; v:v:v:v) in the second one. Plates were sprayed with molybdatophosphoric acid (3.5%; Merck )

for detection of the total polar lipids. PE, phosphatidethanolamine; AL1–6, unknown aminolipids; L1–19, unknown lipids; APL, unknown phospholipids. All data are from

this study. Proﬁles were labelled following the established pattern by Montero-Calasanz et al. [38].

346 M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350

Table 1

Differential phenotypic characteristics of strain CT348 and the type strains of closely related species of the genus Chryseobacterium based on respiration measurements by

TM T T T T

Biolog Phenotype MicroArray technique. Strains: 1, C. oleae CT348 sp. nov.; 2, C. daecheongense DSM 15235 ; 3, C. deﬂuvii DSM 14219 ; 4, C. gambrini DSM 18014 ; 5, C.

T T T T T T

gleum DSM 16776 ; 6, C. gregarium DSM 19109 ; 7, C. indologenes DSM 16777 ; 8, C. jejuense DSM 19299 ; 9, C. joostei DSM 16927 ; 10, C. luteum DSM 18605 ; 11, C. shigense

T T T T T

DSM 17126 ; 12, C. taiwanense JCM 21767 ; 13, C. ureilyticum DSM 18017 ; 14, C. vrystaatense DSM 25206 ; 15, C. wanjuense DSM 17724 . All physiological data are from

this study. +, positive reaction; - negative reaction; +/−, ambiguous.

Characteristics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

d-Trehalose + + + + + − + + + + + - + + +

d-Cellobiose − + + + +/− − − + − − − − +

Sucrose − + +/− + − + − − − + + − − +/− −

−

d-Turanose − + + +/− + − − + − − +/− +/− +/− −

−

Stachyose − +/− +/− − − − − − − − − − +/− −

-Salicin − − − − + + − − − + + − − − −

-Fructose + +/− + + + − + + + + + + +/− +

d-Serine + − +/− − + − + + + + + − + + +/−

d-Mannitol + − − + − − − − − − + − − + −

−

Glycerol + + + + + + + + + + − + + +

d-Fructose-6-phosphate + +/− + +/− − + + − + − +/− +/− + + +

d-Aspartic acid + − − − − − − + − − − − − − −

l-Alanine + + − − + + + + + − + + + + −

l-Arginine + + + + + +/− + + + − + + + +/− +

l-Histidine − − − − − − + − + − − − +/− − −

l-Serine + + + − + + + + + + + + + + −

-Glucuronic acid + +/− +/− − +/− +/− + − − − − − + +/− −

Quinic acid − − − − − + − − − − − − − − −

l-Malic acid + +/− − − − + − − - − − − − +/− −

given in other publications. The work of Herzog et al. [20] implies vrystaatense (97.8%) and C. luteum (97.6%). Furthermore, the strain

that the correct structure should be 9Z, 15-methyl hexadecenoic CT348 and the closest relatives were placed within the same

acid, while the MIDI annotation implies 7Z, 15-methyl hexade- phylogenetic group by both maximum-likelihood and maximum-

cenoic acid. Thus, the description of the genus Chyseobacterium parsimony methods (Fig. 2). The 16S rRNA analysis revealed thus

should be emended to include the fact that the fatty acids pat- that the novel strain belongs to the genus Chryseobacterium and

tern of the genus Chyseobacterium contains, in addition to iso-C15:0, forms a species of its own. In addition DNA–DNA hybridizations to

␻

iso-C17:1 7c (that may be incorrectly annotated) and iso-C17:0 3- prove the genomic distinctness of the Strain CT348 representing

OH, as a major fatty acid together with a fatty acid with an ECL the novel species and the above mentioned closest type species dis-

that corresponds with iso-C15:0 2-OH, that is annotated as part of played a DNA–DNA relatedness of 32.0 ± 1.9% with C. vrystaatense,

summed feature 3. The DNA G + C content was 38.2 mol%. This value and 18.4 ± 0.7% with C. luteum, both values being far below the

is consistent with what has been observed for species of the genus threshold value of 70% recommended by Wayne et al. [60] for

Chryseobacterium [5,58]. placement of a novel strain in either a novel species or a different

The almost complete (1480 bp) 16S rRNA gene sequence of species. These data clearly conﬁrmed the observation reported by

strain CT348 was determined. The 16S rRNA sequences showed Stackebrandt and Ebers [48] and Meier-Kolthoff et al. [33] on such

the highest similarity with the type strains of the species C. strains showing 16S rRNA gene sequence similarities of 97–98%.

Table 2

T T T

Cellular fatty acid proﬁles (%) of strain CT348 and closely related species of the genus Chryseobacterium. Strains: 1, C. oleae CT348 sp. nov.; 2, C. daecheongense DSM 15235 ;

T T T T T T

3, C. deﬂuvii DSM 14219 ; 4, C. gambrini DSM 18014 ; 5, C. gleum DSM 16776 ; 6, C. gregarium DSM 19109 ; 7, C. indologenes DSM 16777 ; 8, C. jejuense DSM 19299 ; 9, C.

T T T T T T

joostei DSM 16927 ; 10, C. luteum DSM 18605 ; 11, C. shigense DSM 17126 ; 12, C. taiwanense JCM 21767 ; 13, C. ureilyticum DSM 18017 ; 14, C. vrystaatense DSM 25206 ;

15, C. wanjuense DSM 17724 . tr, Trace (<1%); ND, not detected; ECL, equivalent chain lenght (i.e. the identity of the fatty acids is unknown). Fatty acids amounting to < 1% of

◦

the total fatty acids in all strains are not shown. All strains were grown on TSBA at 28 C for 24 h. All data are from this study.

Fatty acid 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

iso-C13:0 tr 1.3 3.1 1.6 tr tr tr tr 1.0 1.0 1.6 tr tr tr tr

ECL 13.565 ND 6.9 6.4 7.8 2.8 4.8 5.0 ND ND 7.6 2.9 4.2 3.0 ND 9.6

iso-C15:0 44.4 48.8 54.8 51.7 35.7 34.6 34.50 36.2 41.4 37.7 41.7 44.5 32.5 42.8 43.1

anteiso-C15:0 tr 2.0 2.7 1.3 tr 8.2 tr tr tr 3.8 tr tr tr 1.2 tr

iso-C16:0 tr tr tr tr tr tr tr tr ND tr tr ND ND tr ND

C16:00 tr 1.2 1.3 1.4 1.3 tr 1.1 1.4 1.1 1.1 1.0 tr 1.1 tr 1.2

iso-C15:0 3-OH 3.2 2.3 2.2 2.1 2.8 1.9 2.3 3.0 2.7 2.1 2.4 3.1 2.9 2.8 2.8

iso-C 17:1 ␻9c 20.4 7.9 5.3 8.0 23.6 21.0 25.5 23.7 23.7 21.2 17.8 16.2 28.2 22.4 11.4

ECL 16.582 ND 1.6 1.3 1.7 1.7 1.3 7 ND ND 1.6 1.8 1.6 1.6 ND 1.7

iso-C17:0 1.1 2.5 2.4 2.2 1.5 tr 1.0 1.4 1.4 tr 1.0 1.3 tr tr 2.2

C16:0 3-OH tr tr tr tr tr tr tr 1.2 tr tr 1.0 tr 1.0 tr tr

iso-C17:0 3-OH 16.4 13.3 10.8 11.5 15.1 8.6 12.7 18.3 15.6 10.6 16.3 17.1 14.4 15.3 13.1

C17:0 2-OH tr tr tr ND ND 1.7 tr tr tr tr tr ND tr tr ND

Summed features

3 (iso-C15:0 2-OH) 8.9 9.5 6.8 8.3 11.6 10.7 11.3 11.2 9.6 7.2 9.9 8.4 10.2 9.2 10.9

4 tr ND ND ND tr tr 1.0 tr tr tr tr ND tr 1.0 tr

Summed features are groups of two or three fatty acids that are treated together for the purpose of evaluation in the MIDI system and include both peaks with discrete

ECLs as well as those where the ECLs are not reported separately. Summed feature 3 listed as iso-C15:0 2-OH/C16:1␻7c and annotated here as iso-C15:0 2-OH on the basis of the

ECL; Summed feature 4 listed as anteiso-C17:1 B and/or iso-C17:1 I (iI).

This fatty acid may be incorrectly identiﬁed, see text for details and is recorded as 9Z, 15-methyl hexadecenoic acid for C. deﬂuvii, C. daecheongense, C. gambrini, C. joostei

and C. ureilyticum by Herzog et al. [20].

M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350 347

Fig. 2. Maximum likelihood phylogenetic tree inferred from 16S rRNA gene sequences, showing the phylogenetic position of strain CT348 relative to the type strains

within the genus Chryseobacterium. The branches are scaled in terms of the expected number of substitutions per site. Support values from maximum-likelihood (left) and

maximum-parsimony (right) bootstrapping are shown above the branches if equal to or larger than 60%. Numbers in parentheses are the number of sequences in each of the

collapsed branches.

Several phenotypic characteristics apart from the phylogenetic branched-chain fatty acids are iso-C15:0, iso-C17:1␻7c (that may be

analysis based on 16S rRNA gene sequences also support the incorrectly annotated in the previous work), iso-C17:0 3-OH and iso-

distinctiveness of strain CT348 from all other Chryseobacterium C15:0 2-OH (annotated as part of summed feature 3; See Table 2,

species (Table 1). Based on the phenotypic and genotypic data pre- but may also be annotated as summed feature 4, depending on the

sented above, we propose that strain CT348 represents a novel version of the MIDI system and the peak naming tables used).

species within the genus Chryseobacterium, and names C. oleae sp.

nov. Emended description of C. daecheongense Kim et al. 2005

Based on a review of the literature and new data obtained in this

study, emended descriptions of the genus Chryseobacterium and The properties are as given in the species description by

the species C. daecheongense, C. gambrini, C. jejuense, C. joostei, C. Kim et al. [28] with the following emendation. In addition to

luteum, C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense phosphatidylethanolamine, the polar lipid proﬁle contains ten

were also provided. unidentiﬁed polar lipids (six lipids and four aminolipids, the Rf val-

ues of which are documented in Fig. 1b). Phosphosphingolipids are

Emended description of the genus Chryseobacterium absent. In addition to the cellular fatty acids listed by Kim et al. [28]

Vandamme et al. 1994 emend. Kämpfer et al. 2009 and Wu the fatty acids labelled as summed feature 4 (iso-C15: 0 2-OH and/or

␻

et al. 2013 C16: 1 7c/t) consist of only iso-C15:0 2-OH, as well as an uniden-

tiﬁed fatty acid (ECL 13.565), smaller amounts of anteiso-C15:0,

␻

The properties are as given by Vandamme et al. [58] and iso-C15:0 3-OH and iso-C17:0. The annotation of iso-C17:1 9c may be

␻

emended by Kämpfer et al. [25] and Wu et al. [63] with the following incorrect and appears to be iso-C17:1 7c. MK-6 is the predominant

emendations. In addition to phosphatidylethanolamine, the polar menaquinone, but MK-5 is present as a minor component.

lipid proﬁle contains three common unknown lipids and two com-

mon unknown aminolipids (Fig. 1). Vandamme et al. [58] report Emended description of C. gambrini Herzog et al. 2008

␻

that the major fatty acids are iso-C15:O, iso-C17:l 9c, iso-C17:O 3-

OH, and summed feature 4 (iso-C15:0 2-OH or C16:1␻7t or both), The properties are as given in the species description by Her-

with the annotation coming from an unspeciﬁed version of the zog et al. [20] with the following emendation. In addition to

MIDI system. While the designation of summed feature 4 (iso-C15:0 phosphatidylethanolamine, the polar lipid proﬁle contains six-

2-OH or C16:1␻7t or both) is clearly different from the annota- teen unidentiﬁed polar lipids (eight lipids, six aminolipids and

tion of summed feature 3 (iso-C15:0 2-OH or C16:1␻7c or both as an aminophospholipid, the Rf values of which are documented in

well as C16:1␻6c or C16:1␻7c or both) earlier work and subsequent Fig. 1c). Phosphosphingolipids are absent. Apart from the fatty acids

mass spectrometric studies indicate that iso-C15:0 2-OH is present listed by Herzog et al. [20] (which are given in IUPAC nomencla-

and C16:1␻7t/C16:1␻6c/C16:1␻7c are absent. Similarly, the fatty ture), cellular fatty acids also consist of an unidentiﬁed fatty acid

acid identiﬁed as iso-C17:1␻9c (which more recent version of the (ECL 13.565) and, small amounts of iso-C17:0 and iso-C15:0 3-OH.

MIDI system may report as summed feature 9 = iso-C17:1␻9c/C16:0 MK-6 is the predominant menaquinone, but MK-5 is present as a

10-methyl) appears to be iso-C17:1␻7c. Consequently the major minor component.

348 M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350

Emended description of C. gleum Holmes et al. 1984 comb to phosphatidylethanolamine, the polar lipid proﬁle contains ten

nov. Vandamme et al. 1994 emend. Nguyen et al. 2013 unidentiﬁed polar lipids (six lipids and four aminolipids, the Rf val-

ues of which are documented in Fig. 1h). Phosphosphingolipids are

The properties are as given in the species description by absent. Apart from fatty acids listed by Shimomura et al. [47], the

Holmes et al. [21] comb nov. Vandamme et al. [58] emend. fatty acids labelled as summed feature 4 (iso-C15: 0 2-OH and/or

␻

Nguyen et al. [40] with the following emendation. In addition to C16: 1 7c/t) consist of only iso-C15:0 2-OH together with small

phosphatidylethanolamine, the polar lipid proﬁle contains twelve amounts of an unidentiﬁed fatty acid (ECL 13.565) and iso-C15:0 3-

unidentiﬁed polar lipids (eight lipids and four aminolipids, the OH. The annotation of iso-C17:1␻9c may be incorrect and appears to

Rf values of which are documented in Fig. 1d). Phosphosphin- be iso-C17:1␻7c. MK-6 is the predominant menaquinone, but MK-5

golipids are absent. Apart from fatty acids listed by Vandamme et is present as a minor component.

al. [58], cellular fatty acids also contain iso-C15:0 2-OH (annotated

as summed feature 4; iso-C 2-OH or C ␻7t or both) together

15:0 16:1 Emended description of C. taiwanense Tai et al. 2006 emend.

with small amounts of an unidentiﬁed fatty acid (ECL 13.565) and

Wu et al. 2013

iso-C15:0 3-OH. The annotation of iso-C17:1␻9c may be incorrect and

appears to be iso-C ␻7c. MK-6 is the predominant menaquinone,

17:1 The properties are as given in the species description by Tai et al.

but MK-5 is present as a minor component.

[52] emend. Wu et al. [63] with the following emendation. In addi-

tion to phosphatidylethanolamine, the polar lipid proﬁle contains

Emended description of C. jejuense Weon et al. 2008 emend. eleven unidentiﬁed polar lipids (seven lipids and four aminolipids,

Wu et al. 2013 the Rf values of which are documented in Fig. 1i). Phosphosph-

ingolipids are absent. Apart from fatty acids listed by Tai et al.

The properties are as given in the species description by Weon [52], the fatty acids labelled as summed feature 3 (iso-C15: 0 2-OH

et al. [61] emend. Wu et al. [63] with the following emendation. and/or C16:1␻7c) consist of only iso-C15:0 2-OH, together with small

In addition to phosphatidylethanolamine, the polar lipid proﬁle amounts of an unidentiﬁed fatty acid (ECL 13.565) and iso-C15:0 3-

contains fourteen unidentiﬁed polar lipids (ten lipids and four OH. The annotation of iso-C17:1␻9c may be incorrect and appears

aminolipids, the Rf values of which are documented in Fig. 1e). to be iso-C17:1␻7c.

Phosphosphingolipids are absent. Apart from fatty acids listed by

Weon et al. [61], cellular fatty acids also consist of iso-C 2-OH

15:0 Emended description of C. ureilyticum Herzog et al. 2008

(annotated as summed feature 3: C16:1␻7c and/or iso-C15:0 2-OH in

the supplementary table to the paper by [61]), together with small

The properties are as given in the species description by Her-

amount of iso-C 3-OH. The annotation of iso-C ␻9c may be

15:0 17:1 zog et al. [20] with the following emendation. In addition to

incorrect and appears to be iso-C ␻7c. MK-6 is the predominant

17:1 phosphatidylethanolamine, the polar lipid proﬁle contains twelve

menaquinone, but MK-5 is present as a minor component.

unidentiﬁed polar lipids (eight lipids and four aminolipids, the Rf

values of which are documented in Fig. 1j). Phosphosphingolipids

Emended description of C. joostei Hugo et al. 2003 are absent. Apart from fatty acids listed by Herzog et al. [20] (which

are given in IUPAC nomenclature), cellular fatty acids also consist

The properties are as given in the species description by Hugo of small amounts of an unidentiﬁed fatty acid (ECL 13.565) and iso-

et al. [22] with the following emendation. In addition to phos- C15:0 3-OH. MK-6 is the predominant menaquinone but MK-5 is

phatidylethanolamine, the polar lipid proﬁle contains fourteen present as a minor component.

unidentiﬁed polar lipids (nine lipids and ﬁve aminolipids, the Rf

values of which are documented in Fig. 1f). Phosphosphingolipids

Emended description of C. vrystaatense de Beer et al. 2005

are absent. Apart from fatty acids listed by Hugo et al. [22], the

fatty acids labelled as summed feature 4 (iso-C 2-OH and/or

15: 0 The properties are as given in the species description by de

␻

C 7c/t) consist of only iso-C 2-OH together with a small

16: 1 15:0 Beer et al. [11] with the following emendation. In addition to

amount of iso-C 3-OH. The annotation of iso-C ␻9c may be

15:0 17:1 phosphatidylethanolamine, the polar lipid proﬁle contains eleven

incorrect and appears to be iso-C ␻7c. MK-6 is the predominant

17:1 unidentiﬁed polar lipids (seven lipids and four aminolipids, the

menaquinone, but MK-5 is present as a minor component.

Rf values of which are documented in Fig. 1k). Phosphosphin-

golipids are absent. Apart from fatty acids listed by de Beer et

Emended description of C. luteum Behrendt et al. 2007 al. [11], the fatty acids labelled as summed feature 3 (iso-C15:0 2-

OH and/or C16:1␻7c) consist of only iso-C15:0 2-OH, together with

The properties are as given in the species description by small amount of iso-C15:0 3-OH. The annotation of iso-C17:1␻9c

Behrendt et al. [3] with the following emendation. In addition to may be incorrect and appears to be iso-C17:1␻7c. MK-6 is the only

phosphatidylethanolamine, the polar lipid proﬁle contains eleven menaquinone.

unidentiﬁed polar lipids (eight lipids and three aminolipids, the Rf

values of which are documented in Fig. 1 g). Phosphosphingolipids

Description of C. oleae sp. nov.

are absent. Apart from fatty acids listed by Behrendt et al. [3], cellu-

lar fatty acids also consist of an unidentiﬁed fatty acid (ECL 13.565),

C. oleae o.le.a e L. gen. fem. oleae referring to olive tree (O.

small amount of anteiso-C and iso-C 3-OH. The annotation

15:0 15:0 europaea L.), the plant from which the type strain was isolated.

of iso-C ␻9c may be incorrect and appears to be iso-C ␻7c.

17:1 17:1 On TSB agar colonies are circular with a shiny surface and

MK-6 is the predominant menaquinone, but MK-5 is present as a

entire edges and produce a yellow to orange non-diffusible

minor component.

ﬂexirubin type pigment. Cells are Gram-staining-negative, aer-

obic, unable to grow anaerobically with nitrate and nitrite as

Emended description of C. shigense Shimomura et al. 2005 terminal electron acceptors, non-spore-forming, non-motile, cata-

lase and oxidase positive and Voges–Proskauer and methyl red

The properties are as given in the species description by Shi- reaction negative. Utilises dextrin, d-maltose, d-trehalose, ␤-

momura et al. [47] with the following emendation. In addition gentiobiose, d-glucose, d-mannose, d-fructose, d- and l-fucose,

M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350 349

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glycyl- -proline, -arginine, -alanine, - and -serine, guanidine sp. nov., Chryseobacterium lathyri sp. nov. and Chryseobacterium rhizosphaerae

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␤ d

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␣

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␣

-mannosidase and ␣-fucosidase activities are absent. NaCl tol- [15] Halebian, S., Harris, B., Finegold, S.M., Rolfei, R.D. (1981) Rapid method that

aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria.

erance ranges from 0 to 1% (w/v). Growth occurs from 5 to

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35 C and pH 5.0 to 8.0. The predominant menaquinone is MK-

[16] Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor

6 but a signiﬁcant amount of MK-5 is also present. The major and analysis program for Windows 95/98/NTL. In: Nucleic Acids Symposium

Series 41, pp. 95–98.

polyamine is sym-homospermidine but minor amounts of sper-

[17] Hamana, K., Matsuzaki, S. (1990) Occurrence of homospermidine as a major

midine and spermine are also present. The main polar lipids

polyamine in the authentic genus Flavobacterium. Can. J. Microbiol. 26,

are phosphatidylethanolamine, three unknown aminolipids and 228–230.

[18] Hamana, K., Itoh, T., Benno, Y., Hayashi, H. (2008) Polyamine distribution pro-

six unknown lipids (the Rf values of which are documented in

ﬁles of the new members of the phylum Bacteroidetes. J. Gen. Appl. Microbiol.

Fig. 1a). Phosphosphingolipids are absent. Cellular fatty acids con-

54, 229–236.

␻

sist mainly of iso-C15:0, C17:1 9c, iso-C17:0 3-OH and iso-C15:0 2-OH, [19] Hantsis-Zacharov, E., Shakéd, T., Senderovich, Y., Halpern, M. (2008) Chry-

seobacterium oranimense sp. nov., a psychrotolerant, proteolytic and lipolytic

with smaller amounts of iso-C15:0 3-OH. The annotation of iso-

bacterium isolated from raw cow’s milk. Int. J. Syst. Evol. Microbiol. 58,

C ␻9c may be incorrect and appears to be iso-C ␻7c. The type

17:1 17:1 2635–2639.

strain has a genomic DNA G + C content of 38.2 mol%. [20] Herzog, P., Winkler, I., Wolking, D., Kämpfer, P., Lipski, A. (2008) Chry-

The type strain, CT348 (= DSM 25575 =CCUG 63020), was iso- seobacterium ureilyticum sp. nov., Chryseobacterium gambrini sp. nov.,

Chryseobacterium pallidum sp. nov. and Chryseobacterium molle sp. nov., isolated

lated in 2006 from the ectorhizosphere of an organic olive tree

from beer-bottling plants. Int. J. Syst. Evol. Microbiol. 58, 26–33.

in Spain in Mairena del Aljarafe (Seville), Spain. The INSDC acces-

[21] Holmes, B., Owen, R.J., Steigerwalt, A.G., Brenner, D.J. (1984) Flavobacterium

sion number for the 16S rRNA gene sequence of strain CT348 is gleum, a new species found in human clinical specimens. Int. J. Syst. Bacteriol.

EU336939. 34, 21–25.

[22] Hugo, C.J., Segers, P., Hoste, B., Vancanneyt, M., Kersters, K. (2003) Chryseobac-

terium joostei sp. nov., isolated from the dairy environment. Int. J. Syst. Evol.

Acknowledgements Microbiol. 53, 771–777.

[23] Huss, V.A.R., Fest, H., Schleifer, K.H. (1983) Studies on the spectrophotometric

determination of DNA hybridization from renaturation rates. Syst. Appl. Micro-

We would like to gratefully acknowledge the help of Bettina

biol. 4, 184–192.

Sträubler and Birgit Grün (DSMZ, Braunschweig), for their help [24] Kämpfer, P., Dreyer, U., Neef, A., Dott, W., Busse, H.-J. (2003) Chryseobacterium

deﬂuvii sp. nov., isolated from wastewater. Int. J. Syst. Evol. Microbiol. 53,

in DNA–DNA hybridisation analysis. M.C. Montero-Calasanz is the

93–97.

recipient of a postdoctoral contract from European Social Fund

[25] Kämpfer, P., Vaneechoutte, M., Lodders, N., De Baere, T., Avesani, V., Janssens,

Operational Programme (2007–2013) for Andalusia and a DSMZ M., Busse, H.-J., Wauters, G. (2009) Description of Chryseobacterium anthropi sp.

nov. to accomodate clinical isolates biochemically similar to Kaistella koreen-

postdoctoral scholarship.

sis and Chryseobacterium haifense, proposal to reclassify Kaistella koreensis as

Chryseobacterium koreense comb. nov. and emended description of the genus

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