Antonie van Leeuwenhoek (2015) 107:759–771 DOI 10.1007/s10482-014-0370-6
ORIGINAL PAPER
Oricola cellulosilytica gen. nov., sp. nov., a cellulosedegrading bacterium of the family Phyllobacteriaceae isolated from surface seashore water, and emended descriptions of Mesorhizobium loti and Phyllobacterium myrsinacearum Asif Hameed • Mariyam Shahina • Wei-An Lai • Shih-Yao Lin • Li-Sen Young • You-Cheng Liu • Yi-Han Hsu • Chiu-Chung Young Received: 14 August 2014 / Accepted: 23 December 2014 / Published online: 8 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain negative, strictly aerobic, rod-shaped, non-spore-forming, motile cellulolytic bacterium, designated strain CC-AMH-0T, was isolated from surface seashore water of Hualien, Taiwan and subjected to polyphasic taxonomy. Strain CC-AMH-0T exhibited enzymatic saccharification of cellulose and active growth particularly during log-phase under nutrient-limited conditions, whereas enhanced saccharification was found in the declining growth phase under
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpD and recA gene sequences of strain CC-AMH-0T are KF582604, KP142843 and KP142844, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0370-6) contains supplementary material, which is available to authorized users. A. Hameed M. Shahina W.-A. Lai S.-Y. Lin Y.-C. Liu Y.-H. Hsu C.-C. Young (&) Department of Soil & Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung 402, Taiwan e-mail: [email protected] L.-S. Young Department of Biotechnology, National Formosa University, No. 64, Wunhua Rd., Huwei Township, Yunlin County 632, Taiwan C.-C. Young Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan
copiotrophic conditions. The novel strain shared high pairwise 16S rRNA gene sequence similarities to Mesorhizobium loti USDA 3471T (96.2 %), Phyllobacterium myrsinacearum IAM 13584T (95.9 %), Hoeflea marina LMG 128T (94.0 %) and other Phyllobacteriaceae members. However, phylogenetic analyses based on 16S rRNA, atpD and recA gene sequences clearly distinguished strain CC-AMH-0T from other representatives of related genera. In addition, strain CC-AMH-0T was distinguished from the above mentioned species by significantly lacking phosphatidylcholine besides accommodating major amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sulfoquinovosyldiacylglycerol; moderate amounts of phosphatidylethanolamine and trace amounts of an unidentified phospholipid, an unidentified aminolipid and an unidentified phosphoglycolipid. Strain CC-AMH-0T possessed C18:1 x7c and/or C18:1 x6c (summed feature 8) as predominant fatty acids, 63.3 mol% DNA G?C content and ubiquinone-10 (Q-10) as the sole respiratory quinone. On the basis of polyphasic taxonomic evidences, strain CC-AMH-0T is proposed to represent a novel genus and species of the family Phyllobacteriaceae, for which the name Oricola cellulosilytica gen. nov., sp. nov. is proposed. The type strain of the type species is CCAMH-0T (=JCM 19534T =BCRC 80694T). Emended descriptions of M. loti and P. myrsinacearum are also proposed.
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Keywords Planctonic protein Cellulosedegrading Phyllobacteriaceae Reducing sugar Carboxymethylcellulose Sulfoquinovosyldiacylglycerol
Introduction The family Phyllobacteriaceae (Kno¨sel 1984) is affiliated to the order Rhizobiales, class Alphaproteobacteria. At the time of writing, the family comprised eleven genera: Phyllobacterium (Kno¨sel 1984; Mergaert et al. 2002), Aminobacter (Urakami et al. 1992), Mesorhizobium (Jarvis et al. 1997), Aquamicrobium (Bambauer et al. 1998; Fritsche et al. 1999; Ka¨mpfer et al. 2009), Pseudaminobacter (Ka¨mpfer et al. 1999), Nitratireductor (Labbe´ et al. 2004), Hoeflea (Peix et al. 2005), Chelativorans (Doronina et al. 2010), ‘Aliihoeflea’ (Roh et al. 2008), Pseudahrensia (Jung et al. 2012) and Thermovum (Yabe et al. 2012). The type species of the genera Pseudahrensia, Nitratireductor, Hoeflea and ‘Aliihoeflea’ were isolated and described from marine environments, whereas the type species of the other genera were originated from non-marine resources. Cells of Phyllobacteriaceae are usually Gramnegative and aerobic rods, which may be motile by means of polar or subpolar or lateral flagella. Phyllobacteriaceae strains are characterized to be rich in straight-chain mono-unsaturated acid C18:1. In addition, they possess ubiquinone-10 (Q-10) as the major respiratory quinone and high ([50.0 mol%) DNA G?C content (Yabe et al. 2012). Polar lipids such as diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), phosphatidyldimethylethanolamine (PDE), hydroxyphosphatidylethanolamine (OH-PME), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) have been reported from Phyllobacteriaceae (Doronina et al. 2010; Ka¨mpfer et al. 1999; Peix et al. 2005; Jung et al. 2012; Yabe et al. 2012). The type species of the genera Phyllobacterium, Aquamicrobium, Nitratireductor, Pseudahrensia and Thermovum exhibit nitrate reduction (Mergaert et al. 2002; Bambauer et al. 1998; Labbe´ et al. 2004; Yabe et al. 2012; Jung et al. 2012), Aquamicrobium can metabolize sulfonated aromatic compounds (Bambauer et al. 1998), whereas Chelativorans,
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Aminobacter and Pseudaminobacter can degrade EDTA, methylamine and salicylate, respectively (Doronina et al. 2010; Urakami et al. 1992; Ka¨mpfer et al. 1999). On the other hand, the type species of the genera Mesorhizobium and Phyllobacterium are plantassociated, in which Mesorhizobium loti can even nodulate leguminous plants (Jarvis et al. 1997; Kno¨sel 1984; Mergaert et al. 2002). However, the ecological relevance of many Phyllobacteriaceae representatives, particularly with regard to the metabolism of naturally occurring high molecular weight (HMW) biopolymers remains largely elusive. Cellulose is a HMW macromolecule abundant in the environment. Several cellulolytic bacterial taxa have been characterized so far from various environmental sources (http://www.wzw.tum.de/mbiotec/ cellmo.htm) including water (Klippel et al. 2011) and soil (Wirth and Ulrich 2002; Woo et al. 2014). Cellulolytic microorganisms or their cellulases are of significant commercial interest particularly in the field of paper, textile and lignocellulosic bioethanol production industries (Acharya and Chaudhary 2012; Cannella and Jørgensen 2014). Cellulolytic microorganisms participate in biogeochemical cycling of carbon by mineralizing the lignocellulosic plant substances. The vast diversity of microorganisms present in cellulolytic environments and the inability of most of the cellulolytic strains to grow under laboratory conditions are considered to be major constraints in understanding microbial cellulolysis in situ (Wilson 2011). Here, we describe the isolation and polyphasic taxonomic characterization of a novel cellulosedegrading marine bacterial strain designated CCAMH-0T, affiliated to the family Phyllobacteriaceae.
Materials and methods Isolation of novel strain, storage, reference strains and culture conditions Strain CC-AMH-0T was isolated from a seawater sample collected near coastal Hualien, Taiwan (24.307512°N 120.518572°E). The marine water sample was subjected to standard dilution-to-extinction plating method using marine agar 2216 (MA; BD Difco) and incubated at 30 °C for 48–96 h. Strain CCAMH-0T appeared as a non-pigmented colony, which was isolated, purified and preserved in marine broth
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(MB) supplemented with 20 % glycerol at -80 °C. Investigations were carried out according to the taxonomic guidelines (Tindall et al. 2010). The type strains of the type species of the genera Phyllobacterium (P. myrsinacearum JCM 20932T =IAM 13584T), Mesorhizobium (M. loti BCRC 80381T =USDA 3471T) and Hoeflea (H. marina LMG 128T) were used for direct comparative taxonomic analysis. Estimation of cell density, planctonic reducing sugar, planctonic protein and cellulase activity Freshly prepared (48 h old) cells of strain CC-AMH0T were inoculated (0.4 %, v/v) into 1 % (w/v) CMC (Sigma) supplemented 50 ml full-strength (MB ? 1 % CMC) and 50 ml half-strength marine broth (MB ? 1 % CMC) and incubated at 30 °C (130 rpm) for about one week. Same volume of noninoculated MB ? 1 % CMC and MB ? 1 % CMC, maintained under similar conditions were served as controls. Culture experiments were performed in triplicate. One ml of each sample was aseptically withdrawn every day and aliquots were directly used for the determination of OD at 600 nm. Remaining aliquots were centrifuged at 8,0009g for 5 min at 4 °C, cell-free culture supernatants were stored at 4 °C and used for subsequent determination of specific activity of cellulase and quantification of planctonic reducing sugar and protein. Planctonic reducing sugar was determined using dinitrosalicylic acid (DNS) reagent (Miller 1959) as follows: 100 ll cell-free culture supernatant was mixed with 400 ll 50 mM sodium phosphate buffer (pH 7.5) and 500 ll DNS reagent and immediately boiled at 100 °C in a water bath for 10 min. Finally, the reaction mixture was diluted fivefold by adding 4 ml of deionized water and absorbance at 540 nm was recorded by using a UV–Vis spectrophotometer. The reducing sugar was estimated using a standard curve plotted for glucose (Sigma). Planctonic proteins were quantified using 15-fold diluted cell-free culture supernatant through Bradford method (Bradford 1976) using protein assay dye reagent concentrate (Microplate method, Bio-Rad; catalog number 500-0006) and bovine serum albumin as a standard. Cellulase activity was estimated according to earlier descriptions (Ghose 1987) with slight modifications. Briefly, 400 ll of 1 % (w/v) CMC (solubilized in 50 mM sodium phosphate buffer, pH 7.5) was mixed with 100 ll cell-free
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culture supernatant and incubated for 30 min at 30 °C. This mixture was subsequently mixed with 500 ll DNS and immediately boiled at 100 °C for 10 min and finally diluted fivefold by adding 4 ml deionized water. The absorbance was spectrophotometrically recorded at 450 nm. The reducing sugar liberated was quantified using a standard curve plotted for glucose. Heat-inactivated (100 °C, 10 min) cell-free culture supernatant channeled through similar process were served as background control. The data were presented as specific activity, which was defined as enzyme activity (IU) per mg protein. One international unit (IU) enzyme activity was defined as the amount of enzyme required to liberate 1 lmol reducing sugar (equivalent to glucose) from CMC per minute under the assay conditions. Statistical analysis Statistical analyses were performed using SPSS software package (version 11.5, SPSS, Inc.). One-way analysis of variance (ANOVA) was used for analyzing each set of data. The data were presented as the mean value of triplicates with standard deviation. A p value of \0.05 (according to Duncan’s multiple range test) was considered as significant. Molecular systematics The genomic DNA was isolated by using the UltraCleanTM Microbial Genomic DNA Isolation Kit (MO BIO, USA) by following the manufacturer’s instructions. The partial 16S rRNA gene was amplified via PCR as described earlier (Shahina et al. 2013). The partial sequences of genes encoding for ATP synthase b-subunit (atpD) and recombinase A (recA) were determined according to Gaunt et al. (2001). Gene sequencing was performed by using the BigDye terminator kit (Heiner et al. 1998) and an automatic DNA sequencer (ABI PRISM 310, Applied Biosystems, CA, USA) (Watts and MacBeath 2001). 16S rRNA gene sequence fragments were assembled using the Fragment Assembly System program from the Wisconsin Package (GCG 1995). Sequence similarity values were computed using BLAST program (Altschul et al. 1990) at National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi) and EzTaxon-e (http://www.ezbiocloud. net/eztaxon) server (Kim et al. 2012). Sequence data
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were analyzed by MEGA 5 (Molecular Evolutionary Genetics Analysis, version 5.0; Tamura et al. 2011), after multiple alignment by Clustal_X (Thompson et al. 1997). Distance matrix method (distance options according to the Kimura two-parameter model; Kimura 1980) including clustering by neighbor-joining (Saitou and Nei 1987), a discrete character-based maximumparsimony (Fitch 1971) and maximum likelihood (Felsenstein 1981) methods were used. The topologies of the trees were evaluated by using the bootstrap resampling method based on 1,000 replications (Felsenstein 1985).
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(NA, Himedia), tryptic soy agar (TSA, BD Difco) and R2A (BD Difco) agar. Activity of catalase and oxidase, and hydrolysis of starch, egg yolk, Tweens 20 and 80, L-tyrosine, casein (skim milk), colloidal chitin, CMC, xylan and DNA were tested as described previously (Hameed et al. 2013); MA and R2A agar were used as basal media for hydrolytic assay of novel strain and reference strains, respectively. Strains were inoculated in API 20 NE and API ZYM strips according to the manufacturers’ instructions. Cell suspensions were prepared in sterile 0.9 % NaCl solution for inoculation.
Phenotypic, physiological and biochemical analyses
Chemotaxonomic analyses
The following phenotypic tests were carried out exclusively on the novel strain. Colonies were examined for morphological features such as colony appearance, size, shape, texture and pigmentation. Presence of endospores was assessed by phasecontrast microscopy (model A3000, Zeiss) after malachite-green staining (Smibert and Krieg 1994) of the cells grown on MA for 7 days. Cell morphology including presence of flagella was determined by placing the cells (1–2 days old) on a carbon-coated copper grid followed by staining with 0.2 % uranyl acetate for 5–10 s, brief air-drying and observation under a transmission electron microscope (JEOL JEM-1400). Gram staining was performed according to Murray et al. (1994). The requirement for NaCl was tested on R2A agar (BD Difco) supplemented with 0–10 % (w/v) NaCl (at 1 % intervals). The pH range (4.0–10.0, at 1.0 pH unit intervals) for growth was determined in MB that was prepared using 100 mM acetate (pH 4–5), 100 mM NaH2PO4/Na2HPO4 (pH 6–8) and 100 mM NaHCO3/ Na2CO3 (pH 9–10) buffers. Growth at 10, 20, 25, 30, 37, 40, 45, 50 and 55 °C was tested in MB after 72 h of incubation. Anaerobic growth was tested using MA or MA supplemented with 0.1 % (w/v) KNO3 by incubating the culture plates in an anaerobic chamber (COY, USA). The strain was inoculated into API 50 CH strip (bioMe´rieux) and Biolog GN2 MicroPlate according to the manufacturers’ instructions. Cell suspension was prepared in sterile 0.9 % NaCl solution for inoculation. The following phenotypic tests were carried out on all four strains. Growth was tested on nutrient agar
Fatty acid methyl esters of all four strains were extracted from cells cultivated on R2A agar supplemented with 1.5 % NaCl (except for M. loti, for which unsupplemented R2A agar was used) at 30 °C for 2–3 days. Cell samples were harvested during exponential growth phase, and subjected to saponification, methylation and extraction as described previously (Ka¨mpfer and Kroppenstedt 1996) followed by gas chromatography (model 7890A, Agilent). Peaks were automatically integrated, and fatty acid names and percentages were determined using the microbial identification standard software package MIDI (version 6) (Sasser 1990) and the database RTSBA6. Respiratory quinones of strain CC-AMH-0T were extracted according to earlier descriptions (Collins 1985; Minnikin et al. 1984) with minor modifications (Shahina et al. 2013). Polar lipids of strain CC-AMH0T were extracted and analyzed by two-dimensional (2-D) thin layer chromatography (TLC) (Embley and Wait 1994); 10 % ethanolic molybdatophosphoric acid, Zinzadze reagent, 0.2 % ninhydrin and a-naphthol were used as reagents to detect the spots corresponding to total lipids, phospholipids, aminolipids and glycolipids, respectively. The TLC spots of PC and SQDG were identified as described earlier (Hameed et al. 2014). In order to determine DNA G?C content, the genomic DNA of strain CC-AMH0T was subjected to thermal denaturation followed by enzymatic digestion into nucleosides as described previously (Mesbah et al. 1989). The resultant nucleoside mixture was separated and quantified by RPHPLC (Mesbah et al. 1989) with minor modifications (Shahina et al. 2013).
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Fig. 1 Cell density (dotted line) and planctonic reducing sugar (solid line) in full-strength (MB, filled circle) and half-strength (MB, open circle) marine broth supplemented with 1 % (w/v) carboxymethylcellulose each. Cells of strain CC-AMH-0T
(0.4 %, v/v) were inoculated at 0 h. Different letters indicate statististically significant differences (p \ 0.05) according to Duncan’s multiple range test; uppercase and lowercase letters correspond to half-strength and full-strength media respectively
Results and discussion
of cellulase during the period of final incubation (142–163 h). Cells showed good growth under the copiotrophic (MB ? 1 % CMC) condition as compared to nutrientlimited (MB ? 1 % CMC) condition in liquid culture. However, under nutrient-limited conditions, cells swiftly attained log phase probably due to active secretion of cellulase, which converted some amounts of CMC into free sugar. This free sugar was presumably consumed for biosynthetic purpose as we detected very low-level of planctonic reducing sugar irrespective of active secretion of cellulase. In addition, significantly low amounts of planctonic protein detected particularly in this phase further corroborated this assumption. On the other hand, detection of significantly high amount of planctonic protein in the later declining growth phase under nutrient limited condition probably indicates cell death and decay. Nonetheless, enzymatic saccharification and efficient conversion of extracellular free sugar and protein into biomass can be hypothesized to be one of the adaptive strategies exhibited by strain CC-AMH-0T to thrive in nutrient-limited/ oligotropic surface seawater. The amplified 16S rRNA gene of strain CC-AMH0T contained 1,455 nucleotides (GenBank/EMBL/
Growth curves obtained for MB ? 1 % CMC and MB ? 1 % CMC showed an almost-similar trend (Fig. 1). However, overall cell density was higher in MB ? 1 % CMC than in MB ? 1 % CMC, reaching maxima at 74 h in both treatments. Cells quickly attained log phase in MB ? 1 % CMC but exhibited significant decline in cell density particularly during the period of final incubation. Planctonic reducing sugar was significantly higher at 74 h in MB ? 1 % CMC and showed an increasing trend during declining growth phase, whereas planctonic reducing sugar was found to be diminished during final incubation period in MB ? 1 % CMC (Fig. 1). Planctonic protein was higher but statistically similar in MB ? 1 % CMC as compared to MB ? 1 % CMC and both treatments showed an almost-similar trend (Fig. 2). However, planctonic protein was significantly higher in declining growth phase and significantly lower during log phase in MB ? 1 % CMC. Specific activity of cellulase was significantly higher during log phase in MB ? 1 % CMC, whereas it was significantly higher during the initial declining phase in MB ? 1 % CMC (Fig. 2). Both treatments showed diminished specific activity
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Fig. 2 Planctonic protein (dotted line) and specific activity of cellulase (solid line) in full-strength (filled circle) and halfstrength (open circle) marine broth supplemented with 1 % (w/ v) carboxymethylcellulose each. Cells of strain CC-AMH-0T
(0.4 %, v/v) were inoculated at 0 h. Different letters indicate statististically significant differences (p \ 0.05) according to Duncan’s multiple range test; uppercase and lowercase letters correspond to half-strength and full-strength media respectively T
Aquamicrobium defluvii DSM 11603 (Y15403) T
Pseudaminobacter salicylatoxidans BN12 (AF072542) T
Nitratireductor lucknowense IITR-21 (HQ658355) T
Mesorhizobium loti LMG 6125 (X67229) 86
T
Aminobacter aminovorans DSM7048 (AJ011759) T
Chelativorans multitrophicus DSM 9103 (EF457243) T
Thermovum composti Nis3 (AB563785) T
Hoeflea marina LMG 128 (AY598817) 86
T
‘Aliihoeflea aestuarii’ N8 (EF660756) T
Phyllobacterium myrsinacearum IAM 13584 (D12789) T
Oricola cellulosilytica CC-AMH-0 (KF582604) T
Pseudahrensia aquimaris DW-32 (GU575117) T
Alteromonas macleodii DSM 6062 (Y18228) 0.02
Fig. 3 Maximum likelihood tree based on 16S rRNA gene sequences showing the phylogenetic relationship between CCAMH-0T and the type species of other representative genera of the family Phyllobacteriaceae. Alteromonas macleodii DSM
6062T (Y18228) was used as an outgroup. Bootstrap values ([70 %) based on 1,000 replications are shown at the nodes. Bar 0.02 substitutions per nucleotide position
DDBJ accession number KF582604). The sequence analysis revealed that strain CC-AMH-0T shared high pairwise similarity to the type strains of the type species of M. loti USDA 3471T (96.2 %), P. myrsinacearum IAM 13584T (95.9 %), ‘Aliihoeflea aestuarii’
N8T (95.5 %), Aquamicrobium defluvii DSM 11603T (95.4 %), Chelativorans multitrophicus DSM 9103T (95.3 %), Pseudaminobacter salicylatoxidans BN12T (95.3 %), Aminobacter aminovorans DSM 7048T (95.0 %), Nitratireductor aquibiodomus NL21T
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(94.9 %), H. marina LMG 128T (94.3 %), Pseudahrensia aquimaris HDW-32T (94.3 %) and Thermovum composti Nis3T (93.9 %). In addition, strain CC-AMH-0T also shared high similarity to Mesorhizobium qingshengii CCBAU 33460T (96.7 %), several other Mesorhizobium species (96.7–96.2 %) and Hoeflea alexandrii AM1V30T (96.3 %). In the maximum likelihood phylogenetic tree that was constructed by exclusively incorporating type species of 11 established Phyllobacteriaceae genera, strain CC-AMH-0T formed a distinct taxonomic lineage (Fig. 3). Similarly, in the neighbor-joining, maximum-parsimony and maximum likelihood phylogenetic trees, which were generated after including some additional taxa, strain CC-AMH-0T further formed a unique phyletic lineage distantly associated with the above mentioned species (Figs. S1, S2, S3). The taxonomic status of strain CC-AMH-0T was further assessed using two house-keeping genes (atpD and recA). Both atpD and recA fragments were amplified from strain CC-AMH-0T (KP142843 and KP142844, respectively), whereas only recA fragment was amplified from H. marina LMG 128T (KP142845). In the maximum likelihood tree based on atpD and recA gene sequences, strain CC-AMH-0T formed a distinct phylogenetic lineage distantly associated with Mesorhizobium species (Figs. S4, S5). Phylogentetic distinctiveness of strain CC-AMH-0T was also conserved in neighbor-joining and maximum likelihood trees (data not shown). Thus, based on the cumulative phylogenetic data of 16S rRNA, atpD and recA gene sequences, strain CC-AMH-0T was appeared to represent a possible novel genus. The morphological features of strain CC-AMH-0T are shown in Fig. S6 and other features are given in the genus and species descriptions. Cells of strain CCAMH-0T were observed to be small rod-to-oval shaped with rounded ends. Cells exhibited motility by means of one or two lateral flagella. The additional characteristics that distinguish the new isolate from its phylogenetic neighbors are given in Table 1. The fatty acid profiles of the strains are summarized in Table 2. Strain CC-AMH-0T was characterized to have predominant amounts of C18:1 x7c and/or C18:1 x6c (summed feature 8; 81.2 %) followed by C18:0 (6.7). Significant amounts of C18:1 x7c and/or C18:1 x6c were found also in M. loti BCRC 80381T and P. myrsinacearum JCM 20932T. On the other hand, the
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branched saturated fatty acid iso-C 16:0 and unsaturated fatty acid C16:1 x5c were found exclusively in H. marina LMG 128T, where as a hydroxyl fatty acid C16:0 3-OH was found only in P. myrsinacearum JCM 20932T. In addition, fatty acids such as C16:0, C17:0, C18:0, C17:1 x8c, C18:1 x7c 11-metyl and C19:0 cyclo x8c exhibited significant quantitative variations in all four strains. Nonetheless, major fatty acids in all strains were in line with the data reported earlier from Phyllobacteriaceae representatives (Yabe et al. 2012). The total polar lipids of strain CC-AMH-0T and three reference strains are shown in Fig. 4 and summarized in Table 1. Strain CC-AMH-0T was found to contain major amounts of DPG, PG, PE and SQDG; moderate amounts of PE and trace amounts of an unidentified phospholipid (PL1), an unidentified aminolipid (AL1) and an unidentified phosphoglycolipid (PGL). DPG, PG and PME were found in all three strains, whereas PE, SQDG and PGL were found only in novel strain and H. marina LMG 128T. Our polar lipid data of H. marina LMG 128T are in excellent agreement with the data reported earlier (Peix et al. 2005; Stevenson et al. 2011). P. myrsinacearum JCM 20932T found to accommodate maximum number of unidentified aminolipids (AL1–4), in which AL2 and AL4 stained orange (Fig. S7). Strain CC-AMH-0T strikingly lacked PC that was found in all three reference strains. In addition, all four strains showed remarkable qualitative/quantitative variations particularly with regard to three unidentified phospholipids (PL1–3) and four unidentified aminolipids (AL1–4). Taken together, the present polar lipid data clearly distinguished strain CC-AMH-0T from other three reference strains. The DNA G?C content of strain CC-AMH-0T was 63.3 mol%, a value within the range reported from M. loti but slightly above than that of P. myrsinacearum and significantly above than that of H. marina (Table 1). High G?C content is one of the characteristic features of Phyllobacteriaceae representatives (Yabe et al. 2012). Ubiquinone-10 (Q-10) was detected as the sole respiratory quinone in strain CC-AMH-0T. Phyllobacteriaceae representatives have been characterized to produce Q-10 as the predominant (Doronina et al. 2010; Jung et al. 2012; Peix et al. 2005; Roh et al. 2008; Urakami et al. 1992; Yabe et al. 2012) or sole respiratory quinone (Stevenson et al. 2011).
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123 ?
R2A
R2A ? 1.5 % NaCl
? w w ? w w w -
Urease
Alkaline phosphatase
Lipase C14
Valine arylamidase
Cystine arylamidase
Trypsin
a-Chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
a-Glucosidase b-Glucosidase
-
Starch
-
L-Arabinose
D-Mannose,
N-acetyl-glucosamine, malic acid, trisodium citrate
-
D-Glucose, D-mannitol, D-maltose
Assimilation of
-
–(DP–)
L-Tyrosine
?
?
PNPG
Tween 20, tween 80
-
Gelatin
CMC, egg yolk
-
Esculin
Hydrolysis of
?
Catalase, oxidase
Activity of
-
-
Reduction of nitrate to nitrite
?
TSA NA
1
MA
Growth at
Characteristic
-
-
-
-
-
w
–(DP–)
-
-
-
? w
w
?
-
-
-
-
-
w
-
-
-
w
?
w -
-
2
?
w
?
?
-
?
?(DP–)
-
-
?
? -
w
?
?
?
?
w
-
?
?
?
-
?
?
? ?
-
3
Table 1 Differential characteristics of novel strain and some of the related type species of the family Phyllobacteriaceae
-
-
?
-
-
-
–(DP–)
?
?
w
? ?
?
?
w
-
-
w
w
?
-
?
?
?
?
? ?
?
4
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Thus, based on the phylogenetic distinctiveness accompanied by chemotaxonomic (including the polar lipid, fatty acid, quinone profiles), phenotypic and a number of biochemical features, the cellulolytic strain CC-AMH-0T is proposed to represent a novel genus and species of the family Phyllobacteriaceae, for which the name Oricola cellulosilytica gen. nov., sp. nov. is proposed. In addition, on the basis of new data obtained in this study, emended descriptions of the species M. loti and P. myrsinacearum are also proposed.
Emended description of Mesorhizobium loti Jarvis et al. 1997 The description is according to Jarvis et al. (1997) with the following amendments. The polar lipids include major amounts PG, PC and an unidentified aminolipid (AL1) and moderate amounts of DPG, PME and an unidentified phospholipid (PL2). In addition, trace amounts of an unidentified phospholipid (PL1) is also present.
Emended description of Phyllobacterium myrsinacearum (ex Kno¨sel 1962) Kno¨sel 1984 The description is according to Kno¨sel (1984) with the following amendments. The polar lipids include major amounts of DPG, PG, PC, PME and an unidentified lipid (AL4). In addition, moderate amounts of three unidentified aminolipids (AL1–3) and an unidentified phospholipid (PL3) are also present.
Mergaert et al. (2002)
Peix et al. (2005) c
b
Jarvis et al. (1997)
Description of Oricola gen. nov
a
PNPG p-nitrophenyl-b-D-galactopyranoside, CMC carboxymethylcellulose, DP diffusible brown-pigments on L-tyrosine agar; Abbreviations for polar lipids are defined in Fig. 4; ? positive, - negative, w weak reaction
Taxa: 1, strain CC-AMH-0T; 2, Mesorhizobium loti BCRC 80381T; 3, Phyllobacterium myrsinacearum JCM 20932T; 4, Hoeflea marina LMG 128T. All data are from this study except indicated otherwise. All strains are positive for activity of esterase (C 4), esterase lipase (C 8), and leucine arylamidase. All strains are negative for the followings: hydrolysis of DNA, casein, colloidal chitin and xylan; indole production and fermentation of glucose; activity of arginine dihydrolase, a-galactosidase, b-galactosidase, bglucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase; assimilation of capric acid and phenylacetic acid
60.3–61.3b 59–64a DNA G?C content (mol%)
63.3
53.1c
DPG, PG, PC, PME, PE, SQDG, AL1, PGL DPG, PG, PC, PME, PL3, AL1–4 DPG, PG, PC, PME, PL1–2, AL1 DPG, PG, PME, PE, SQDG, PL1, AL1, PGL Total polar lipids
? ? w Potassium gluconate Adipic acid
Characteristic
Table 1 continued
1
4 3 2
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Oricola (o.ri’ co.la. L. fem n. ora coastline, L. masc. suffix -cola inhabitant of. N.L. masc. n. oricola inhabitant of the coast). Cells are Gram-stain negative, strictly aerobic, nonspore forming, chemoheterotrophic, mesophilic, rodto-oval shaped with rounded ends; occasionally appear slightly curved and motile by means of one or two lateral flagella. Catalase- and oxidase- positive. The predominant fatty acid is C18:1 x7c and/or C18:1 x6c (summed feature 8). The major polar lipids are
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Table 2 Whole cell fatty acid profile (%) of members of the family Phyllobacteriaceae Fatty acid
1
2
3
4
C16:0
2.3
C17:0 C18:0
TR 6.7
10.6
5.1
1.7
1 2.3
TR 1.9
6.9 TR
iso-C14:0 iso-C16:0
–
–
–
1
–
–
–
24.1
iso-C17:0
–
4.8
–
1.6
iso-C18:0
–
–
–
1.8
Saturated
Branched saturated
Unsaturated C15:1 x5c
–
–
–
1.3
C16:1 x5c
–
–
–
9.1
C17:1 x8c
–
1
TR
17.7
C18:1 x9c
–
1
–
2.5
C18:1 x7c 11-metyl
2.7
13.9
3.9
–
iso-C16:1 H
–
–
–
1.4
iso-C17:1 x10c
TR
–
1
1.3
Hydroxy C16:0 3-OH
–
–
6.2
–
C18:0 3-OH
–
–
1.7
–
C18:1 2-OH
–
TR
2.6
–
C19:0 cyclo x8c
2.8
7.4
14.9
–
Summed feature 2
–
–
4.5
–
Summed feature 3
TR
1.1
1.4
1.5
Summed feature 6
–
–
–
1.4
Summed feature 8
81.2
53.5
51.8
23.0
Branched monounsaturated
Cyclo
As indicated by Montero-Calasanz et al. (2013) 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 equivalent chain lengths (ECL) as well as those where the ECL are not reported separately. Summed feature 2 was listed as C14:0 3-OH and/or iso-C16:1 I; Summed feature 3 was listed as C16:1 x6c and/or C16:1 x7c; Summed feature 6 was listed as C19:1 x11c and/or C19:1 x9c; Summed feature 8 was listed as C18:1 x7c and/or C18:1 x6c Taxa: 1, strain CC-AMH-0T; 2, Mesorhizobium loti BCRC 80381T; 3, Phyllobacterium myrsinacearum JCM 20932T; 4, Hoeflea marina LMG 128T. All data are from this study. Major ([5 %) components are shown in bold type. The fatty acids amounting to \1.0 % of the total fatty acids in all strains are not shown. TR trace (\1.0 %), – not detected
DPG, PG, PME and SQDG. In addition, a moderate amount of PE is also present. Ubiquinone-10 (Q-10) is the sole respiratory quinone. As determined by 16S rRNA gene sequence analysis, the genus Oricola is affiliated to the family Phyllobacteriaceae. The type species is Oricola cellulosilytica.
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Description of Oricola cellulosilytica sp. nov Oricola cellulosilytica (ce.llu.lo.si.ly.ti.ca. N.L. n. cellulosum cellulose; Gr. adj. lytikos dissolving; N.L. adj. lytica dissolving; N.L. fem. adj. cellulosilytica cellulose-dissolving).
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Fig. 4 Polar lipid profiles of strain CC-AMH-0T (a), Mesorhizobium loti BCRC 80381T (b), Phyllobacterium myrsinacearum JCM 20932T (c) and Hoeflea marina LMG 128T (d) as determined by two-dimensional thin layer chromatography. The total polar lipids were visualized by spraying the TLC plates with 10 % ethanolic
molybdatophosphoric acid. PE phosphatidylethanolamine, PME phosphatidylmonomethylethanolamine, DPG diphosphatidylglycerol, PG phosphatidylglycerol, PC phosphatidylcholine, SQDG sulfoquinovosyldiacylglycerol, PGL phosphoglycolipid, AL1–4 unidentified aminolipids, PL1–3 unidentified phospholipids
Cells are rod-shaped, 0.7–0.9 lm in diameter and 1.1–2.3 lm in length. On MA, after 1–2 days of incubation at 30 °C colonies are circular, convex and non-pigmented, 0.5–1.0 mm in diameter. Growth occurs at 20–40 °C (optimum, 25–30 °C), at pH 6.0–10.0 (optimum, 7.0–8.0) and in the presence of 0–7 % NaCl (optimum, 1–3 %). Growth occurs on MA but not on NA, TSA and R2A agar. Catalase- and oxidase- positive. Egg yolk and CMC are hydrolyzed, whereas L-tyrosine, starch, Tweens 20 and 80, chitin, casein, xylan and DNA are not hydrolyzed. In the GN2 Biolog MicroPlate, positive for the oxidation of
following substrates: glycogen, Tween 80, D-fructose, a-D-glucose, b-methyl-D-glucoside, L-rhamnose, formic acid and glycyl-L-glutamic acid; the remaining substrates are not oxidized. In the API 20 NE strip, positive for hydrolysis of p-nitrophenyl-b-D-galactopyranoside; negative for nitrate reduction, indole production, D-glucose fermentation, arginine dihydrolase and urease activities, hydrolysis of gelatin and esculin, and assimilation of D-glucose, L-arabinose, Dmannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. In
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the API ZYM strip, positive for alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and trypsin activities; weakly positive for valine arylamidase, cystine arylamidase, a-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities; and negative for lipase (C 14), agalactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities. In the API 50 CH strip, acid is produced from D-galactose, Dglucose, D-fructose and potassium 5-ketogluconate. In addition to the major fatty acid listed above, significant amounts of C18:0 is also present. In addition to the polar lipids listed above, an unidentified phospholipid (PL1), an unidentified aminolipid (AL1) and an unidentified phosphoglycolipid (PGL) are present in trace amounts. The DNA G?C content of the type strain is 63.3 mol%. The type strain CC-AMH-0T (=JCM 19534T =BCRC 80694T) was isolated from surface seawater off coastal Hualien, Taiwan. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpD and recA gene sequences of strain CC-AMH-0T are KF582604, KP142843 and KP142844, respectively. Acknowledgments We would like to thank Prof. Hans G. Tru¨per for nomenclatural advice and etymology. This work was supported in part by the Ministry of Education, Taiwan, R.O.C. under the ATU plan. We acknowledge Cheng-Zhe Wen and YuMing Huang for technical assistance.
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ORIGINAL PAPER
Oricola cellulosilytica gen. nov., sp. nov., a cellulosedegrading bacterium of the family Phyllobacteriaceae isolated from surface seashore water, and emended descriptions of Mesorhizobium loti and Phyllobacterium myrsinacearum Asif Hameed • Mariyam Shahina • Wei-An Lai • Shih-Yao Lin • Li-Sen Young • You-Cheng Liu • Yi-Han Hsu • Chiu-Chung Young Received: 14 August 2014 / Accepted: 23 December 2014 / Published online: 8 January 2015 Ó Springer International Publishing Switzerland 2015
Abstract A Gram-stain negative, strictly aerobic, rod-shaped, non-spore-forming, motile cellulolytic bacterium, designated strain CC-AMH-0T, was isolated from surface seashore water of Hualien, Taiwan and subjected to polyphasic taxonomy. Strain CC-AMH-0T exhibited enzymatic saccharification of cellulose and active growth particularly during log-phase under nutrient-limited conditions, whereas enhanced saccharification was found in the declining growth phase under
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpD and recA gene sequences of strain CC-AMH-0T are KF582604, KP142843 and KP142844, respectively.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0370-6) contains supplementary material, which is available to authorized users. A. Hameed M. Shahina W.-A. Lai S.-Y. Lin Y.-C. Liu Y.-H. Hsu C.-C. Young (&) Department of Soil & Environmental Sciences, College of Agriculture and Natural Resources, National Chung Hsing University, Taichung 402, Taiwan e-mail: [email protected] L.-S. Young Department of Biotechnology, National Formosa University, No. 64, Wunhua Rd., Huwei Township, Yunlin County 632, Taiwan C.-C. Young Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan
copiotrophic conditions. The novel strain shared high pairwise 16S rRNA gene sequence similarities to Mesorhizobium loti USDA 3471T (96.2 %), Phyllobacterium myrsinacearum IAM 13584T (95.9 %), Hoeflea marina LMG 128T (94.0 %) and other Phyllobacteriaceae members. However, phylogenetic analyses based on 16S rRNA, atpD and recA gene sequences clearly distinguished strain CC-AMH-0T from other representatives of related genera. In addition, strain CC-AMH-0T was distinguished from the above mentioned species by significantly lacking phosphatidylcholine besides accommodating major amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sulfoquinovosyldiacylglycerol; moderate amounts of phosphatidylethanolamine and trace amounts of an unidentified phospholipid, an unidentified aminolipid and an unidentified phosphoglycolipid. Strain CC-AMH-0T possessed C18:1 x7c and/or C18:1 x6c (summed feature 8) as predominant fatty acids, 63.3 mol% DNA G?C content and ubiquinone-10 (Q-10) as the sole respiratory quinone. On the basis of polyphasic taxonomic evidences, strain CC-AMH-0T is proposed to represent a novel genus and species of the family Phyllobacteriaceae, for which the name Oricola cellulosilytica gen. nov., sp. nov. is proposed. The type strain of the type species is CCAMH-0T (=JCM 19534T =BCRC 80694T). Emended descriptions of M. loti and P. myrsinacearum are also proposed.
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Keywords Planctonic protein Cellulosedegrading Phyllobacteriaceae Reducing sugar Carboxymethylcellulose Sulfoquinovosyldiacylglycerol
Introduction The family Phyllobacteriaceae (Kno¨sel 1984) is affiliated to the order Rhizobiales, class Alphaproteobacteria. At the time of writing, the family comprised eleven genera: Phyllobacterium (Kno¨sel 1984; Mergaert et al. 2002), Aminobacter (Urakami et al. 1992), Mesorhizobium (Jarvis et al. 1997), Aquamicrobium (Bambauer et al. 1998; Fritsche et al. 1999; Ka¨mpfer et al. 2009), Pseudaminobacter (Ka¨mpfer et al. 1999), Nitratireductor (Labbe´ et al. 2004), Hoeflea (Peix et al. 2005), Chelativorans (Doronina et al. 2010), ‘Aliihoeflea’ (Roh et al. 2008), Pseudahrensia (Jung et al. 2012) and Thermovum (Yabe et al. 2012). The type species of the genera Pseudahrensia, Nitratireductor, Hoeflea and ‘Aliihoeflea’ were isolated and described from marine environments, whereas the type species of the other genera were originated from non-marine resources. Cells of Phyllobacteriaceae are usually Gramnegative and aerobic rods, which may be motile by means of polar or subpolar or lateral flagella. Phyllobacteriaceae strains are characterized to be rich in straight-chain mono-unsaturated acid C18:1. In addition, they possess ubiquinone-10 (Q-10) as the major respiratory quinone and high ([50.0 mol%) DNA G?C content (Yabe et al. 2012). Polar lipids such as diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PME), phosphatidyldimethylethanolamine (PDE), hydroxyphosphatidylethanolamine (OH-PME), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) have been reported from Phyllobacteriaceae (Doronina et al. 2010; Ka¨mpfer et al. 1999; Peix et al. 2005; Jung et al. 2012; Yabe et al. 2012). The type species of the genera Phyllobacterium, Aquamicrobium, Nitratireductor, Pseudahrensia and Thermovum exhibit nitrate reduction (Mergaert et al. 2002; Bambauer et al. 1998; Labbe´ et al. 2004; Yabe et al. 2012; Jung et al. 2012), Aquamicrobium can metabolize sulfonated aromatic compounds (Bambauer et al. 1998), whereas Chelativorans,
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Aminobacter and Pseudaminobacter can degrade EDTA, methylamine and salicylate, respectively (Doronina et al. 2010; Urakami et al. 1992; Ka¨mpfer et al. 1999). On the other hand, the type species of the genera Mesorhizobium and Phyllobacterium are plantassociated, in which Mesorhizobium loti can even nodulate leguminous plants (Jarvis et al. 1997; Kno¨sel 1984; Mergaert et al. 2002). However, the ecological relevance of many Phyllobacteriaceae representatives, particularly with regard to the metabolism of naturally occurring high molecular weight (HMW) biopolymers remains largely elusive. Cellulose is a HMW macromolecule abundant in the environment. Several cellulolytic bacterial taxa have been characterized so far from various environmental sources (http://www.wzw.tum.de/mbiotec/ cellmo.htm) including water (Klippel et al. 2011) and soil (Wirth and Ulrich 2002; Woo et al. 2014). Cellulolytic microorganisms or their cellulases are of significant commercial interest particularly in the field of paper, textile and lignocellulosic bioethanol production industries (Acharya and Chaudhary 2012; Cannella and Jørgensen 2014). Cellulolytic microorganisms participate in biogeochemical cycling of carbon by mineralizing the lignocellulosic plant substances. The vast diversity of microorganisms present in cellulolytic environments and the inability of most of the cellulolytic strains to grow under laboratory conditions are considered to be major constraints in understanding microbial cellulolysis in situ (Wilson 2011). Here, we describe the isolation and polyphasic taxonomic characterization of a novel cellulosedegrading marine bacterial strain designated CCAMH-0T, affiliated to the family Phyllobacteriaceae.
Materials and methods Isolation of novel strain, storage, reference strains and culture conditions Strain CC-AMH-0T was isolated from a seawater sample collected near coastal Hualien, Taiwan (24.307512°N 120.518572°E). The marine water sample was subjected to standard dilution-to-extinction plating method using marine agar 2216 (MA; BD Difco) and incubated at 30 °C for 48–96 h. Strain CCAMH-0T appeared as a non-pigmented colony, which was isolated, purified and preserved in marine broth
Antonie van Leeuwenhoek (2015) 107:759–771
(MB) supplemented with 20 % glycerol at -80 °C. Investigations were carried out according to the taxonomic guidelines (Tindall et al. 2010). The type strains of the type species of the genera Phyllobacterium (P. myrsinacearum JCM 20932T =IAM 13584T), Mesorhizobium (M. loti BCRC 80381T =USDA 3471T) and Hoeflea (H. marina LMG 128T) were used for direct comparative taxonomic analysis. Estimation of cell density, planctonic reducing sugar, planctonic protein and cellulase activity Freshly prepared (48 h old) cells of strain CC-AMH0T were inoculated (0.4 %, v/v) into 1 % (w/v) CMC (Sigma) supplemented 50 ml full-strength (MB ? 1 % CMC) and 50 ml half-strength marine broth (MB ? 1 % CMC) and incubated at 30 °C (130 rpm) for about one week. Same volume of noninoculated MB ? 1 % CMC and MB ? 1 % CMC, maintained under similar conditions were served as controls. Culture experiments were performed in triplicate. One ml of each sample was aseptically withdrawn every day and aliquots were directly used for the determination of OD at 600 nm. Remaining aliquots were centrifuged at 8,0009g for 5 min at 4 °C, cell-free culture supernatants were stored at 4 °C and used for subsequent determination of specific activity of cellulase and quantification of planctonic reducing sugar and protein. Planctonic reducing sugar was determined using dinitrosalicylic acid (DNS) reagent (Miller 1959) as follows: 100 ll cell-free culture supernatant was mixed with 400 ll 50 mM sodium phosphate buffer (pH 7.5) and 500 ll DNS reagent and immediately boiled at 100 °C in a water bath for 10 min. Finally, the reaction mixture was diluted fivefold by adding 4 ml of deionized water and absorbance at 540 nm was recorded by using a UV–Vis spectrophotometer. The reducing sugar was estimated using a standard curve plotted for glucose (Sigma). Planctonic proteins were quantified using 15-fold diluted cell-free culture supernatant through Bradford method (Bradford 1976) using protein assay dye reagent concentrate (Microplate method, Bio-Rad; catalog number 500-0006) and bovine serum albumin as a standard. Cellulase activity was estimated according to earlier descriptions (Ghose 1987) with slight modifications. Briefly, 400 ll of 1 % (w/v) CMC (solubilized in 50 mM sodium phosphate buffer, pH 7.5) was mixed with 100 ll cell-free
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culture supernatant and incubated for 30 min at 30 °C. This mixture was subsequently mixed with 500 ll DNS and immediately boiled at 100 °C for 10 min and finally diluted fivefold by adding 4 ml deionized water. The absorbance was spectrophotometrically recorded at 450 nm. The reducing sugar liberated was quantified using a standard curve plotted for glucose. Heat-inactivated (100 °C, 10 min) cell-free culture supernatant channeled through similar process were served as background control. The data were presented as specific activity, which was defined as enzyme activity (IU) per mg protein. One international unit (IU) enzyme activity was defined as the amount of enzyme required to liberate 1 lmol reducing sugar (equivalent to glucose) from CMC per minute under the assay conditions. Statistical analysis Statistical analyses were performed using SPSS software package (version 11.5, SPSS, Inc.). One-way analysis of variance (ANOVA) was used for analyzing each set of data. The data were presented as the mean value of triplicates with standard deviation. A p value of \0.05 (according to Duncan’s multiple range test) was considered as significant. Molecular systematics The genomic DNA was isolated by using the UltraCleanTM Microbial Genomic DNA Isolation Kit (MO BIO, USA) by following the manufacturer’s instructions. The partial 16S rRNA gene was amplified via PCR as described earlier (Shahina et al. 2013). The partial sequences of genes encoding for ATP synthase b-subunit (atpD) and recombinase A (recA) were determined according to Gaunt et al. (2001). Gene sequencing was performed by using the BigDye terminator kit (Heiner et al. 1998) and an automatic DNA sequencer (ABI PRISM 310, Applied Biosystems, CA, USA) (Watts and MacBeath 2001). 16S rRNA gene sequence fragments were assembled using the Fragment Assembly System program from the Wisconsin Package (GCG 1995). Sequence similarity values were computed using BLAST program (Altschul et al. 1990) at National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi) and EzTaxon-e (http://www.ezbiocloud. net/eztaxon) server (Kim et al. 2012). Sequence data
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were analyzed by MEGA 5 (Molecular Evolutionary Genetics Analysis, version 5.0; Tamura et al. 2011), after multiple alignment by Clustal_X (Thompson et al. 1997). Distance matrix method (distance options according to the Kimura two-parameter model; Kimura 1980) including clustering by neighbor-joining (Saitou and Nei 1987), a discrete character-based maximumparsimony (Fitch 1971) and maximum likelihood (Felsenstein 1981) methods were used. The topologies of the trees were evaluated by using the bootstrap resampling method based on 1,000 replications (Felsenstein 1985).
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(NA, Himedia), tryptic soy agar (TSA, BD Difco) and R2A (BD Difco) agar. Activity of catalase and oxidase, and hydrolysis of starch, egg yolk, Tweens 20 and 80, L-tyrosine, casein (skim milk), colloidal chitin, CMC, xylan and DNA were tested as described previously (Hameed et al. 2013); MA and R2A agar were used as basal media for hydrolytic assay of novel strain and reference strains, respectively. Strains were inoculated in API 20 NE and API ZYM strips according to the manufacturers’ instructions. Cell suspensions were prepared in sterile 0.9 % NaCl solution for inoculation.
Phenotypic, physiological and biochemical analyses
Chemotaxonomic analyses
The following phenotypic tests were carried out exclusively on the novel strain. Colonies were examined for morphological features such as colony appearance, size, shape, texture and pigmentation. Presence of endospores was assessed by phasecontrast microscopy (model A3000, Zeiss) after malachite-green staining (Smibert and Krieg 1994) of the cells grown on MA for 7 days. Cell morphology including presence of flagella was determined by placing the cells (1–2 days old) on a carbon-coated copper grid followed by staining with 0.2 % uranyl acetate for 5–10 s, brief air-drying and observation under a transmission electron microscope (JEOL JEM-1400). Gram staining was performed according to Murray et al. (1994). The requirement for NaCl was tested on R2A agar (BD Difco) supplemented with 0–10 % (w/v) NaCl (at 1 % intervals). The pH range (4.0–10.0, at 1.0 pH unit intervals) for growth was determined in MB that was prepared using 100 mM acetate (pH 4–5), 100 mM NaH2PO4/Na2HPO4 (pH 6–8) and 100 mM NaHCO3/ Na2CO3 (pH 9–10) buffers. Growth at 10, 20, 25, 30, 37, 40, 45, 50 and 55 °C was tested in MB after 72 h of incubation. Anaerobic growth was tested using MA or MA supplemented with 0.1 % (w/v) KNO3 by incubating the culture plates in an anaerobic chamber (COY, USA). The strain was inoculated into API 50 CH strip (bioMe´rieux) and Biolog GN2 MicroPlate according to the manufacturers’ instructions. Cell suspension was prepared in sterile 0.9 % NaCl solution for inoculation. The following phenotypic tests were carried out on all four strains. Growth was tested on nutrient agar
Fatty acid methyl esters of all four strains were extracted from cells cultivated on R2A agar supplemented with 1.5 % NaCl (except for M. loti, for which unsupplemented R2A agar was used) at 30 °C for 2–3 days. Cell samples were harvested during exponential growth phase, and subjected to saponification, methylation and extraction as described previously (Ka¨mpfer and Kroppenstedt 1996) followed by gas chromatography (model 7890A, Agilent). Peaks were automatically integrated, and fatty acid names and percentages were determined using the microbial identification standard software package MIDI (version 6) (Sasser 1990) and the database RTSBA6. Respiratory quinones of strain CC-AMH-0T were extracted according to earlier descriptions (Collins 1985; Minnikin et al. 1984) with minor modifications (Shahina et al. 2013). Polar lipids of strain CC-AMH0T were extracted and analyzed by two-dimensional (2-D) thin layer chromatography (TLC) (Embley and Wait 1994); 10 % ethanolic molybdatophosphoric acid, Zinzadze reagent, 0.2 % ninhydrin and a-naphthol were used as reagents to detect the spots corresponding to total lipids, phospholipids, aminolipids and glycolipids, respectively. The TLC spots of PC and SQDG were identified as described earlier (Hameed et al. 2014). In order to determine DNA G?C content, the genomic DNA of strain CC-AMH0T was subjected to thermal denaturation followed by enzymatic digestion into nucleosides as described previously (Mesbah et al. 1989). The resultant nucleoside mixture was separated and quantified by RPHPLC (Mesbah et al. 1989) with minor modifications (Shahina et al. 2013).
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Fig. 1 Cell density (dotted line) and planctonic reducing sugar (solid line) in full-strength (MB, filled circle) and half-strength (MB, open circle) marine broth supplemented with 1 % (w/v) carboxymethylcellulose each. Cells of strain CC-AMH-0T
(0.4 %, v/v) were inoculated at 0 h. Different letters indicate statististically significant differences (p \ 0.05) according to Duncan’s multiple range test; uppercase and lowercase letters correspond to half-strength and full-strength media respectively
Results and discussion
of cellulase during the period of final incubation (142–163 h). Cells showed good growth under the copiotrophic (MB ? 1 % CMC) condition as compared to nutrientlimited (MB ? 1 % CMC) condition in liquid culture. However, under nutrient-limited conditions, cells swiftly attained log phase probably due to active secretion of cellulase, which converted some amounts of CMC into free sugar. This free sugar was presumably consumed for biosynthetic purpose as we detected very low-level of planctonic reducing sugar irrespective of active secretion of cellulase. In addition, significantly low amounts of planctonic protein detected particularly in this phase further corroborated this assumption. On the other hand, detection of significantly high amount of planctonic protein in the later declining growth phase under nutrient limited condition probably indicates cell death and decay. Nonetheless, enzymatic saccharification and efficient conversion of extracellular free sugar and protein into biomass can be hypothesized to be one of the adaptive strategies exhibited by strain CC-AMH-0T to thrive in nutrient-limited/ oligotropic surface seawater. The amplified 16S rRNA gene of strain CC-AMH0T contained 1,455 nucleotides (GenBank/EMBL/
Growth curves obtained for MB ? 1 % CMC and MB ? 1 % CMC showed an almost-similar trend (Fig. 1). However, overall cell density was higher in MB ? 1 % CMC than in MB ? 1 % CMC, reaching maxima at 74 h in both treatments. Cells quickly attained log phase in MB ? 1 % CMC but exhibited significant decline in cell density particularly during the period of final incubation. Planctonic reducing sugar was significantly higher at 74 h in MB ? 1 % CMC and showed an increasing trend during declining growth phase, whereas planctonic reducing sugar was found to be diminished during final incubation period in MB ? 1 % CMC (Fig. 1). Planctonic protein was higher but statistically similar in MB ? 1 % CMC as compared to MB ? 1 % CMC and both treatments showed an almost-similar trend (Fig. 2). However, planctonic protein was significantly higher in declining growth phase and significantly lower during log phase in MB ? 1 % CMC. Specific activity of cellulase was significantly higher during log phase in MB ? 1 % CMC, whereas it was significantly higher during the initial declining phase in MB ? 1 % CMC (Fig. 2). Both treatments showed diminished specific activity
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Fig. 2 Planctonic protein (dotted line) and specific activity of cellulase (solid line) in full-strength (filled circle) and halfstrength (open circle) marine broth supplemented with 1 % (w/ v) carboxymethylcellulose each. Cells of strain CC-AMH-0T
(0.4 %, v/v) were inoculated at 0 h. Different letters indicate statististically significant differences (p \ 0.05) according to Duncan’s multiple range test; uppercase and lowercase letters correspond to half-strength and full-strength media respectively T
Aquamicrobium defluvii DSM 11603 (Y15403) T
Pseudaminobacter salicylatoxidans BN12 (AF072542) T
Nitratireductor lucknowense IITR-21 (HQ658355) T
Mesorhizobium loti LMG 6125 (X67229) 86
T
Aminobacter aminovorans DSM7048 (AJ011759) T
Chelativorans multitrophicus DSM 9103 (EF457243) T
Thermovum composti Nis3 (AB563785) T
Hoeflea marina LMG 128 (AY598817) 86
T
‘Aliihoeflea aestuarii’ N8 (EF660756) T
Phyllobacterium myrsinacearum IAM 13584 (D12789) T
Oricola cellulosilytica CC-AMH-0 (KF582604) T
Pseudahrensia aquimaris DW-32 (GU575117) T
Alteromonas macleodii DSM 6062 (Y18228) 0.02
Fig. 3 Maximum likelihood tree based on 16S rRNA gene sequences showing the phylogenetic relationship between CCAMH-0T and the type species of other representative genera of the family Phyllobacteriaceae. Alteromonas macleodii DSM
6062T (Y18228) was used as an outgroup. Bootstrap values ([70 %) based on 1,000 replications are shown at the nodes. Bar 0.02 substitutions per nucleotide position
DDBJ accession number KF582604). The sequence analysis revealed that strain CC-AMH-0T shared high pairwise similarity to the type strains of the type species of M. loti USDA 3471T (96.2 %), P. myrsinacearum IAM 13584T (95.9 %), ‘Aliihoeflea aestuarii’
N8T (95.5 %), Aquamicrobium defluvii DSM 11603T (95.4 %), Chelativorans multitrophicus DSM 9103T (95.3 %), Pseudaminobacter salicylatoxidans BN12T (95.3 %), Aminobacter aminovorans DSM 7048T (95.0 %), Nitratireductor aquibiodomus NL21T
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(94.9 %), H. marina LMG 128T (94.3 %), Pseudahrensia aquimaris HDW-32T (94.3 %) and Thermovum composti Nis3T (93.9 %). In addition, strain CC-AMH-0T also shared high similarity to Mesorhizobium qingshengii CCBAU 33460T (96.7 %), several other Mesorhizobium species (96.7–96.2 %) and Hoeflea alexandrii AM1V30T (96.3 %). In the maximum likelihood phylogenetic tree that was constructed by exclusively incorporating type species of 11 established Phyllobacteriaceae genera, strain CC-AMH-0T formed a distinct taxonomic lineage (Fig. 3). Similarly, in the neighbor-joining, maximum-parsimony and maximum likelihood phylogenetic trees, which were generated after including some additional taxa, strain CC-AMH-0T further formed a unique phyletic lineage distantly associated with the above mentioned species (Figs. S1, S2, S3). The taxonomic status of strain CC-AMH-0T was further assessed using two house-keeping genes (atpD and recA). Both atpD and recA fragments were amplified from strain CC-AMH-0T (KP142843 and KP142844, respectively), whereas only recA fragment was amplified from H. marina LMG 128T (KP142845). In the maximum likelihood tree based on atpD and recA gene sequences, strain CC-AMH-0T formed a distinct phylogenetic lineage distantly associated with Mesorhizobium species (Figs. S4, S5). Phylogentetic distinctiveness of strain CC-AMH-0T was also conserved in neighbor-joining and maximum likelihood trees (data not shown). Thus, based on the cumulative phylogenetic data of 16S rRNA, atpD and recA gene sequences, strain CC-AMH-0T was appeared to represent a possible novel genus. The morphological features of strain CC-AMH-0T are shown in Fig. S6 and other features are given in the genus and species descriptions. Cells of strain CCAMH-0T were observed to be small rod-to-oval shaped with rounded ends. Cells exhibited motility by means of one or two lateral flagella. The additional characteristics that distinguish the new isolate from its phylogenetic neighbors are given in Table 1. The fatty acid profiles of the strains are summarized in Table 2. Strain CC-AMH-0T was characterized to have predominant amounts of C18:1 x7c and/or C18:1 x6c (summed feature 8; 81.2 %) followed by C18:0 (6.7). Significant amounts of C18:1 x7c and/or C18:1 x6c were found also in M. loti BCRC 80381T and P. myrsinacearum JCM 20932T. On the other hand, the
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branched saturated fatty acid iso-C 16:0 and unsaturated fatty acid C16:1 x5c were found exclusively in H. marina LMG 128T, where as a hydroxyl fatty acid C16:0 3-OH was found only in P. myrsinacearum JCM 20932T. In addition, fatty acids such as C16:0, C17:0, C18:0, C17:1 x8c, C18:1 x7c 11-metyl and C19:0 cyclo x8c exhibited significant quantitative variations in all four strains. Nonetheless, major fatty acids in all strains were in line with the data reported earlier from Phyllobacteriaceae representatives (Yabe et al. 2012). The total polar lipids of strain CC-AMH-0T and three reference strains are shown in Fig. 4 and summarized in Table 1. Strain CC-AMH-0T was found to contain major amounts of DPG, PG, PE and SQDG; moderate amounts of PE and trace amounts of an unidentified phospholipid (PL1), an unidentified aminolipid (AL1) and an unidentified phosphoglycolipid (PGL). DPG, PG and PME were found in all three strains, whereas PE, SQDG and PGL were found only in novel strain and H. marina LMG 128T. Our polar lipid data of H. marina LMG 128T are in excellent agreement with the data reported earlier (Peix et al. 2005; Stevenson et al. 2011). P. myrsinacearum JCM 20932T found to accommodate maximum number of unidentified aminolipids (AL1–4), in which AL2 and AL4 stained orange (Fig. S7). Strain CC-AMH-0T strikingly lacked PC that was found in all three reference strains. In addition, all four strains showed remarkable qualitative/quantitative variations particularly with regard to three unidentified phospholipids (PL1–3) and four unidentified aminolipids (AL1–4). Taken together, the present polar lipid data clearly distinguished strain CC-AMH-0T from other three reference strains. The DNA G?C content of strain CC-AMH-0T was 63.3 mol%, a value within the range reported from M. loti but slightly above than that of P. myrsinacearum and significantly above than that of H. marina (Table 1). High G?C content is one of the characteristic features of Phyllobacteriaceae representatives (Yabe et al. 2012). Ubiquinone-10 (Q-10) was detected as the sole respiratory quinone in strain CC-AMH-0T. Phyllobacteriaceae representatives have been characterized to produce Q-10 as the predominant (Doronina et al. 2010; Jung et al. 2012; Peix et al. 2005; Roh et al. 2008; Urakami et al. 1992; Yabe et al. 2012) or sole respiratory quinone (Stevenson et al. 2011).
123
123 ?
R2A
R2A ? 1.5 % NaCl
? w w ? w w w -
Urease
Alkaline phosphatase
Lipase C14
Valine arylamidase
Cystine arylamidase
Trypsin
a-Chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
a-Glucosidase b-Glucosidase
-
Starch
-
L-Arabinose
D-Mannose,
N-acetyl-glucosamine, malic acid, trisodium citrate
-
D-Glucose, D-mannitol, D-maltose
Assimilation of
-
–(DP–)
L-Tyrosine
?
?
PNPG
Tween 20, tween 80
-
Gelatin
CMC, egg yolk
-
Esculin
Hydrolysis of
?
Catalase, oxidase
Activity of
-
-
Reduction of nitrate to nitrite
?
TSA NA
1
MA
Growth at
Characteristic
-
-
-
-
-
w
–(DP–)
-
-
-
? w
w
?
-
-
-
-
-
w
-
-
-
w
?
w -
-
2
?
w
?
?
-
?
?(DP–)
-
-
?
? -
w
?
?
?
?
w
-
?
?
?
-
?
?
? ?
-
3
Table 1 Differential characteristics of novel strain and some of the related type species of the family Phyllobacteriaceae
-
-
?
-
-
-
–(DP–)
?
?
w
? ?
?
?
w
-
-
w
w
?
-
?
?
?
?
? ?
?
4
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Thus, based on the phylogenetic distinctiveness accompanied by chemotaxonomic (including the polar lipid, fatty acid, quinone profiles), phenotypic and a number of biochemical features, the cellulolytic strain CC-AMH-0T is proposed to represent a novel genus and species of the family Phyllobacteriaceae, for which the name Oricola cellulosilytica gen. nov., sp. nov. is proposed. In addition, on the basis of new data obtained in this study, emended descriptions of the species M. loti and P. myrsinacearum are also proposed.
Emended description of Mesorhizobium loti Jarvis et al. 1997 The description is according to Jarvis et al. (1997) with the following amendments. The polar lipids include major amounts PG, PC and an unidentified aminolipid (AL1) and moderate amounts of DPG, PME and an unidentified phospholipid (PL2). In addition, trace amounts of an unidentified phospholipid (PL1) is also present.
Emended description of Phyllobacterium myrsinacearum (ex Kno¨sel 1962) Kno¨sel 1984 The description is according to Kno¨sel (1984) with the following amendments. The polar lipids include major amounts of DPG, PG, PC, PME and an unidentified lipid (AL4). In addition, moderate amounts of three unidentified aminolipids (AL1–3) and an unidentified phospholipid (PL3) are also present.
Mergaert et al. (2002)
Peix et al. (2005) c
b
Jarvis et al. (1997)
Description of Oricola gen. nov
a
PNPG p-nitrophenyl-b-D-galactopyranoside, CMC carboxymethylcellulose, DP diffusible brown-pigments on L-tyrosine agar; Abbreviations for polar lipids are defined in Fig. 4; ? positive, - negative, w weak reaction
Taxa: 1, strain CC-AMH-0T; 2, Mesorhizobium loti BCRC 80381T; 3, Phyllobacterium myrsinacearum JCM 20932T; 4, Hoeflea marina LMG 128T. All data are from this study except indicated otherwise. All strains are positive for activity of esterase (C 4), esterase lipase (C 8), and leucine arylamidase. All strains are negative for the followings: hydrolysis of DNA, casein, colloidal chitin and xylan; indole production and fermentation of glucose; activity of arginine dihydrolase, a-galactosidase, b-galactosidase, bglucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase; assimilation of capric acid and phenylacetic acid
60.3–61.3b 59–64a DNA G?C content (mol%)
63.3
53.1c
DPG, PG, PC, PME, PE, SQDG, AL1, PGL DPG, PG, PC, PME, PL3, AL1–4 DPG, PG, PC, PME, PL1–2, AL1 DPG, PG, PME, PE, SQDG, PL1, AL1, PGL Total polar lipids
? ? w Potassium gluconate Adipic acid
Characteristic
Table 1 continued
1
4 3 2
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Oricola (o.ri’ co.la. L. fem n. ora coastline, L. masc. suffix -cola inhabitant of. N.L. masc. n. oricola inhabitant of the coast). Cells are Gram-stain negative, strictly aerobic, nonspore forming, chemoheterotrophic, mesophilic, rodto-oval shaped with rounded ends; occasionally appear slightly curved and motile by means of one or two lateral flagella. Catalase- and oxidase- positive. The predominant fatty acid is C18:1 x7c and/or C18:1 x6c (summed feature 8). The major polar lipids are
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Table 2 Whole cell fatty acid profile (%) of members of the family Phyllobacteriaceae Fatty acid
1
2
3
4
C16:0
2.3
C17:0 C18:0
TR 6.7
10.6
5.1
1.7
1 2.3
TR 1.9
6.9 TR
iso-C14:0 iso-C16:0
–
–
–
1
–
–
–
24.1
iso-C17:0
–
4.8
–
1.6
iso-C18:0
–
–
–
1.8
Saturated
Branched saturated
Unsaturated C15:1 x5c
–
–
–
1.3
C16:1 x5c
–
–
–
9.1
C17:1 x8c
–
1
TR
17.7
C18:1 x9c
–
1
–
2.5
C18:1 x7c 11-metyl
2.7
13.9
3.9
–
iso-C16:1 H
–
–
–
1.4
iso-C17:1 x10c
TR
–
1
1.3
Hydroxy C16:0 3-OH
–
–
6.2
–
C18:0 3-OH
–
–
1.7
–
C18:1 2-OH
–
TR
2.6
–
C19:0 cyclo x8c
2.8
7.4
14.9
–
Summed feature 2
–
–
4.5
–
Summed feature 3
TR
1.1
1.4
1.5
Summed feature 6
–
–
–
1.4
Summed feature 8
81.2
53.5
51.8
23.0
Branched monounsaturated
Cyclo
As indicated by Montero-Calasanz et al. (2013) 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 equivalent chain lengths (ECL) as well as those where the ECL are not reported separately. Summed feature 2 was listed as C14:0 3-OH and/or iso-C16:1 I; Summed feature 3 was listed as C16:1 x6c and/or C16:1 x7c; Summed feature 6 was listed as C19:1 x11c and/or C19:1 x9c; Summed feature 8 was listed as C18:1 x7c and/or C18:1 x6c Taxa: 1, strain CC-AMH-0T; 2, Mesorhizobium loti BCRC 80381T; 3, Phyllobacterium myrsinacearum JCM 20932T; 4, Hoeflea marina LMG 128T. All data are from this study. Major ([5 %) components are shown in bold type. The fatty acids amounting to \1.0 % of the total fatty acids in all strains are not shown. TR trace (\1.0 %), – not detected
DPG, PG, PME and SQDG. In addition, a moderate amount of PE is also present. Ubiquinone-10 (Q-10) is the sole respiratory quinone. As determined by 16S rRNA gene sequence analysis, the genus Oricola is affiliated to the family Phyllobacteriaceae. The type species is Oricola cellulosilytica.
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Description of Oricola cellulosilytica sp. nov Oricola cellulosilytica (ce.llu.lo.si.ly.ti.ca. N.L. n. cellulosum cellulose; Gr. adj. lytikos dissolving; N.L. adj. lytica dissolving; N.L. fem. adj. cellulosilytica cellulose-dissolving).
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Fig. 4 Polar lipid profiles of strain CC-AMH-0T (a), Mesorhizobium loti BCRC 80381T (b), Phyllobacterium myrsinacearum JCM 20932T (c) and Hoeflea marina LMG 128T (d) as determined by two-dimensional thin layer chromatography. The total polar lipids were visualized by spraying the TLC plates with 10 % ethanolic
molybdatophosphoric acid. PE phosphatidylethanolamine, PME phosphatidylmonomethylethanolamine, DPG diphosphatidylglycerol, PG phosphatidylglycerol, PC phosphatidylcholine, SQDG sulfoquinovosyldiacylglycerol, PGL phosphoglycolipid, AL1–4 unidentified aminolipids, PL1–3 unidentified phospholipids
Cells are rod-shaped, 0.7–0.9 lm in diameter and 1.1–2.3 lm in length. On MA, after 1–2 days of incubation at 30 °C colonies are circular, convex and non-pigmented, 0.5–1.0 mm in diameter. Growth occurs at 20–40 °C (optimum, 25–30 °C), at pH 6.0–10.0 (optimum, 7.0–8.0) and in the presence of 0–7 % NaCl (optimum, 1–3 %). Growth occurs on MA but not on NA, TSA and R2A agar. Catalase- and oxidase- positive. Egg yolk and CMC are hydrolyzed, whereas L-tyrosine, starch, Tweens 20 and 80, chitin, casein, xylan and DNA are not hydrolyzed. In the GN2 Biolog MicroPlate, positive for the oxidation of
following substrates: glycogen, Tween 80, D-fructose, a-D-glucose, b-methyl-D-glucoside, L-rhamnose, formic acid and glycyl-L-glutamic acid; the remaining substrates are not oxidized. In the API 20 NE strip, positive for hydrolysis of p-nitrophenyl-b-D-galactopyranoside; negative for nitrate reduction, indole production, D-glucose fermentation, arginine dihydrolase and urease activities, hydrolysis of gelatin and esculin, and assimilation of D-glucose, L-arabinose, Dmannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. In
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the API ZYM strip, positive for alkaline phosphatase, esterase (C 4), esterase lipase (C 8), leucine arylamidase and trypsin activities; weakly positive for valine arylamidase, cystine arylamidase, a-chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities; and negative for lipase (C 14), agalactosidase, b-galactosidase, b-glucuronidase, aglucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities. In the API 50 CH strip, acid is produced from D-galactose, Dglucose, D-fructose and potassium 5-ketogluconate. In addition to the major fatty acid listed above, significant amounts of C18:0 is also present. In addition to the polar lipids listed above, an unidentified phospholipid (PL1), an unidentified aminolipid (AL1) and an unidentified phosphoglycolipid (PGL) are present in trace amounts. The DNA G?C content of the type strain is 63.3 mol%. The type strain CC-AMH-0T (=JCM 19534T =BCRC 80694T) was isolated from surface seawater off coastal Hualien, Taiwan. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, atpD and recA gene sequences of strain CC-AMH-0T are KF582604, KP142843 and KP142844, respectively. Acknowledgments We would like to thank Prof. Hans G. Tru¨per for nomenclatural advice and etymology. This work was supported in part by the Ministry of Education, Taiwan, R.O.C. under the ATU plan. We acknowledge Cheng-Zhe Wen and YuMing Huang for technical assistance.
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