Antonie van Leeuwenhoek Journal of Microbiology

Vibrio cortegadensis sp. nov., isolated from clams--Manuscript Draft--

Manuscript Number: ANTO-D-13-00264R3

Full Title: Vibrio cortegadensis sp. nov., isolated from clams

Article Type: Original Research Paper

Keywords: Vibrio; V. cortegadensis sp. nov.; MLSA; DDH

Corresponding Author: Jesús L. Romalde, Ph.D.Univ. Santiago de CompostelaSantiago de Compostela, SPAIN

Corresponding Author SecondaryInformation:

Corresponding Author's Institution: Univ. Santiago de Compostela

Corresponding Author's SecondaryInstitution:

First Author: Aide Lasa, M.Sc.

First Author Secondary Information:

Order of Authors: Aide Lasa, M.Sc.

Ana L Diéguez, M.Sc.

Jesús L. Romalde, Ph.D.

Order of Authors Secondary Information:

Abstract: A group of four strains isolated from clams (Venerupis decussata and V. philippinarum)in Galicia (NW Spain) were subjected to a polyphasic characterization, based on thephenotypic characteristics, the analysis of chemotaxonomic features, the sequencingof the 16S rRNA and five housekeeping (atpA, pyrH, recA, rpoA and rpoD) genes, aswell as DNA-DNA hybridization (DDH). The analysis of the phenotypic andchemotaxonomic characteristics and the results of a phylogenetic study, based on the16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), clearlyindicated that these strains belong to the genus Vibrio and were allocated between theSplendidus and Anguillarum clades showing a close relationship with the type strainsof V. tapetis (98.8%), V. pomeroyi (98.0%) and V. crassostreae (97.9%). DDH resultsconfirmed that these isolates constitute a new species. The name Vibrio cortegadensissp. nov. is proposed with C 16.17T (= CECT 7227T = LMG 27474T) as the type strain.

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Dr. Elena Ivanova

Editor

Antonie van Leeuwenhoek Journal of Microbiology.

Santiago de Compostela, November 11, 2013

Dear Dr. Ivanova,

Thank you very much for the language edition on the ANTO-D-13-00264R2 by Lasa et

al., entitled " Vibrio cortegadensis sp. nov. isolated from clams. We are submitting a

new revised version, which was modified taking into account the suggestions of the

attached file sent.

Sincerely,

Dr. Jesus L. Romalde

*Cover LetterClick here to download Cover Letter: Coverletter.doc

Dr. Elena Ivanova

Editor

Antonie van Leeuwenhoek Journal of Microbiology.

Santiago de Compostela, November 7, 2013

Dear Dr. Ivanova,

Thank you very much for the language edition on the ANTO-D-13-

00264R2 by Lasa et al., entitled " Vibrio cortegadensis sp. nov. isolated

from clams. We are submitting a new revised version, which was modified

taking into account the suggestions of the attached file sent.

Yours sincerely,

Dr. Jesus l. Romalde

*Response to reviewers' commentsClick here to download Response to reviewers' comments: Letter to editor.docx

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Vibrio cortegadensis sp. nov., isolated from clams 4

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Aide Lasa, Ana L. Diéguez, and Jesús L. Romalde* 6

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Departamento de Microbiología y Parasitología, CIBUS. Universidad de Santiago de 8

Compostela. Campus Sur s/n. 15782. Santiago de Compostela. Spain. 9

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Running title: Vibrio cortegadensis sp. nov. 16

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Submitted to: Antoine Van Leeuwenhoek, November 2013 19

Revised Ms. ANTO-D-13-00264R2 20

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26 *Corresponding author: 27

Telf.: (+34) 981 563100 ext. 16961 28

Fax: (+34) 981 528085 29

E–mail: jesus.romalde@usc.es 30

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*ManuscriptClick here to download Manuscript: Lasa et al EI edits 9.11.13_rev.doc Click here to view linked References

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ABSTRACT 32

A group of four strains isolated from clams (Venerupis decussata and V. philippinarum) 33

in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the 34

phenotypic characteristics, the analysis of chemotaxonomic features, the sequencing of 35

the 16S rRNA and five housekeeping (atpA, pyrH, recA, rpoA and rpoD) genes, as 36

well as DNA–DNA hybridization (DDH). The analysis of the phenotypic and 37

chemotaxonomic characteristics and the results of a phylogenetic study, based on the 38

16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA), clearly 39

indicated that these strains belong to the genus Vibrio and were allocated between the 40

Splendidus and Anguillarum clades showing a close relationship with the type strains of 41

V. tapetis (98.8%), V. pomeroyi (98.0%) and V. crassostreae (97.9%). DDH results 42

confirmed that these isolates constitute a new species. The name Vibrio cortegadensis 43

sp. nov. is proposed with C 16.17T (= CECT 7227

T = LMG 27474

T) as the type strain. 44

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Keywords: Vibrio, V. cortegadensis sp. nov., MLSA, DDH 47

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INTRODUCTION 59

60

The genus Vibrio comprises of a large number of species that are common inhabitants 61

of aquatic environments such as estuarine, coastal waters and sediments (Colwell 2006; 62

Thompson and Swings 2006). Several species of this genus have been associated with 63

marine eukaryotic organisms including fish, molluscs and crustaceans (Beaz–Hidalgo et 64

al. 2010) and, in addition, some of them have been described as pathogens to fish, 65

molluscs and crustaceans (Farto et al. 2003; Gay et al. 2004; Gómez–León et al. 2005; 66

Jensen et al. 2003; Kueh and Chan 1985; Lacoste et al. 2001; Le Roux et al. 2002; 67

Leano et al. 1998; Nicolas et al. 1996; Pujalte et al. 1993; Sugumar et al. 1998). 68

The increasing number of environmental studies and the introduction of molecular 69

techniques in bacterial taxonomy, such as DNA–DNA hybridization (DDH), multilocus 70

sequence analysis (MLSA) and amplified fragment length polymorphism (AFLP) 71

(Beaz–Hidalgo et al. 2008; Colwell 2006; Thompson and Swings 2006), have enhanced 72

the understanding of the family Vibrionaceae taxonomic structure and phylogeny. 73

Nowadays, there are 98 validly described species of the genus Vibrio, including two 74

subspecies (http://www.vibriobiology.net), that have been grouped into 14 clades 75

(Sawabe et al. 2007). The large number of the species described in the last 6 years, 76

together with the proposal of the new clades (i.g. Marisflavi and Comitans)(Pujalte, 77

2011), have led to an update of the intra-genus classification (Gomez-Gil, personnal 78

communication). 79

In a previous study on the diversity of vibrios conducted in 2004 and 2005, a collection 80

of isolates were obtained from reared clams, Manila clam (Venerupis philippinarum) 81

and carpet–shell clam (V. decussata), aquacultured in different geographical sites of the 82

coast of Galicia (NW Spain). A representative number of isolates of this collection were 83

analysed by AFLP and a group of four strains (cluster 68) could not be assigned to any 84

of the currently known species of the genus Vibrio (Beaz–Hidalgo et al. 2008). In the 85

present study, a polyphasic approach was employed for the characterization of the 86

cluster of four strains isolated from the clams. 87

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MATERIALS AND METHODS 90

91

Bacterial isolates 92

Bacterial strains C 16.17T (=CECT 7227

T= LMG 27474

T), CMJ 9.12 (=CECT 8125= 93

LMG 27475), CMJ 12.11 (=LMG 27477) and Rd 13.7(= LMG 27476) were 94

corresponded to the clam isolates of cluster 68 of Beaz–Hidalgo et al. (2008). Strain C 95

16.17T was isolated from Ria de Arousa (42º 37´3” N/ 8º 46´ 38” W), strains CMJ 9.12 96

and CMJ 12.11 were isolated from Ria de Camariñas (43º 8´16” N/ 9º 10´37” W) and 97

strain Rd 13.7 was isolated from Ria de Vigo (42º 18´27” N/ 8º 37´13” W). The four 98

isolates were characterized in comparison with the following reference strains obtained 99

from bacterial culture collections: V. atlanticus CECT 7223T, V. artabrorum CECT 100

7226T, V. celticus CECT 7224

T, V. chagasii LMG 21353

T, V. crassostreae CAIM 101

1405T, V. cyclitrophicus LMG 21359

T, V. gallaecicus CECT 7244

T, Vibrio gigantis 102

LMG 22741T, V. kanaloae LMG 20539

T, V. lentus CECT 5110

T, V. pomeroyi LMG 103

20537T, V. splendidus CECT 628

T, V. tasmaniensis LMG 20012

T, V. tapetis CECT 104

4600T

and V. anguillarum ATCC 19264T. All strains were cultured on plates of Marine 105

agar (MA, Difco) at 24±1 ºC for 24 h. Stock cultures were maintained frozen at –80 ºC 106

in Marine broth (MB, Pronadisa, Spain) supplemented with 15% of glycerol (v/v). 107

108

Phenotypical characterization 109

The four marine strains were subjected to the following phenotypic tests (MacFaddin 110

1993; Romalde and Toranzo 1991): cell morphology and motility, Gram stain, oxidase, 111

catalase, oxidation/fermentation test, fermentation and acid production from inositol, 112

mannitol and sucrose, gas and acid production from glucose, indole, methyl red, Voges–113

Proskauer reaction, utilization of citrate, arginine dihydrolase test (Moeller’s medium), 114

lysine and ornithine decarboxylation (Moeller’s medium), nitrate reduction, hydrolysis 115

of gelatin, Tween 80, amylase and aesculin. Salt tolerance test was performed on Basal 116

medium agar (BMA, neopeptone [4 g/l], yeast extract [1 g/l], bacteriological agar [15 117

g/l]) supplemented with 0, 0.5, 1, 3, 6, 8 and 10% NaCl. Growth at different 118

temperatures (4, 20, 25, 30, 37 and 44 ºC), pH (4-10), and on thiosulfate–citrate–bile 119

sucrose (TCBS) agar (Oxoid) were also determined. Sensitivity to the vibriostatic agent 120

O/129 (2,4–diamino–6,7–diisopropylpteridine) (150 µg per disc) was determined on 121

Müeller–Hinton (Oxoid) agar. All media were supplemented with 1% NaCl when 122

required. 123

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Additional phenotypic characteristics were performed using API 20 NE, API 50CH and 124

API ZYM miniaturized systems (BioMerieux, France) using Saline solution (SS, 0.85% 125

NaCl) to prepare the bacterial suspensions. API 50CH was used with the slight 126

modifications described by Prado et al. (2005). Briefly, bacterial suspensions were 127

prepared in SS, adjusted to an OD580 of 1.0 and mixed (1:10, v/v) with ZOF medium 128

(without agar) (Lemos et al. 1985) for the inoculation of the strips. With the exception 129

of the growth at different temperatures, all phenotypic tests were performed at 24±1 ºC. 130

131

16S rRNA and housekeeping genes sequencing 132

Genomic DNA for sequencing was obtained as described previously (Osorio et al. 133

1999). Amplification and sequencing of the 16S rRNA gene and the housekeeping 134

genes atpA (ATP synthase alpha subunit gene), recA (recombinase A gene), pyrH 135

(uridine monophosphate kinase gene), rpoA (RNA polymerase alpha subunit gene) and 136

rpoD (RNA polymerase sigma factor gene) were performed according to Thompson et 137

al. (2004, 2005, 2007) and Pascual et al. (2010). For reference strains, sequences were 138

acquired from GenBank/EMBL/DDBJ. Sequence analyses were performed using the 139

DNASTAR Lasergene SEQMAN program. Sequence similarities of 16S rRNA and 140

housekeeping genes were determined using the EzTaxon-e server (www.eztaxon-141

e.ezbiocloud.net; Kim et al. 2012) and the BLASTN program respectively. Sequences 142

were aligned using CLUSTAL W tool (Larkin et al. 2007), and phylogenetic trees were 143

reconstructed using the neighbour-joining and maximum-likelihood algorithms (MEGA 144

version 5.05) (Tamura et al. 2011). Distance matrices were calculated by using 145

Kimura’s two-parameter correction and stability of the groupings was estimated by 146

bootstrap analysis (1000 replicates) using the MEGA version 5.0 (Tamura et al., 2011). 147

148

DNA–DNA hybridization (DDH) 149

Genomic DNA for DDH experiments was extracted using the commercial DNeasy 150

Blood & Tissue kit (QIAGEN), following the manufacturer´s protocol. DDH 151

experiments were undertaken between the strain C16.17T and the type strains of the 152

species with highest similarities in the 16S rRNA gene (V. tapetis, V. pomeroyi and V. 153

crassostreae) and a representative of the Anguillarum clade (V. anguillarum). DDH 154

experiments were performed with the hydroxyapatite/microtitre plate method (Ziemke 155

et al. 1998) using a hybridization temperature (Tm) of 60 ºC. Reciprocal reactions (i.e. 156

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A×B and B×A) were performed and were generally within the limits of this method 157

(Goris et al. 1998). 158

159

Fatty acids analysis 160

Chemotaxonomic features were studied by the analyses of fatty acid methyl esters 161

(FAME). FAME were extracted and prepared from 24 h cultures on MA incubated at 162

24±1ºC as described by Sasser et al. (1990) according to the MIDI Microbial 163

Identifications System (MIDI, Newark, DE, USA). The two closest species in the 164

MLSA were analysed in parallel for comparison. 165

166

RESULTS AND DISCUSSION 167

168

The four marine strains were facultative anaerobic, motile, Gram–negative rods. 169

Positive for oxidase production and reduction of nitrates to nitrites, only one of the 170

strains (CMJ 12.11) was positive in the catalase test. They required salt for growth 171

(optimal range 1-3%), were able to grow at 4 ºC but not at 37 or 44ºC (optimal range 172

20-25ºC). Optimal pH was found to be in the range of 6 to 9. The strains C 16.17T and 173

CMJ 9.12 grew on thiosulfate–citrate–bile–sucrose agar (TCBS, Oxoid) but not the 174

strains CMJ 12.11 and Rd 13.7. They were sensitive to the vibriostatic agent O/129. 175

Differentiating phenotypic features for the four marine strains are shown in the Table 176

S1. 177

Sequence similarity of the 16S rRNA gene indicated that the isolates belonged to the 178

genus Vibrio. Isolate C16.17T showed highest sequence similarities with the species V. 179

tapetis (98.7%), V. pomeroyi (98.0%), and V. crassostreae (97.9%). Phylogenetic 180

analysis based on 16S rRNA gene sequences of the isolates, employing both NJ or ML 181

approaches, showed that the four marine strains present an intermediate position 182

between the representatives of Splendidus and Anguillarum clades (Figures 1 and S1). 183

Multilocus sequence analysis (MLSA) of housekeeping genes has been proposed as a 184

useful tool to define the phylogenetic relationships among microorganisms 185

(Stackebrandt and Ebers, 2006). In the genus Vibrio, several genes have been studied 186

for delineating new species, such as gyrB, atpA, recA, pyrH or dnaJ (Pascual et al. 187

2010; Sawabe et al. 2007; Thompson et al. 2004, 2005, 2007). In this study, sequences 188

of the genes atpA (1300 bp), pyrH (575 bp), recA (765 bp), rpoA (875 bp) and rpoD 189

(842 bp) were obtained for the clam isolates and compared with the closest relatives. 190

7

Each housekeeping gene pointed different species of the genus Vibrio as the closest 191

relative, with similarity values lower than 93% in all cases (Figure S2). Phylogenetic 192

trees based on each housekeeping gene and on concatenated sequences of the five 193

housekeeping genes, not only showed that the four clam isolates form a tight group, but 194

also suggested a closer relationship with the Splendidus clade (Figures 2, S2 and S3). 195

Further studies will confirm the inclusion of this group of isolates in a specific clade of 196

the genus. The GenBank accession numbers for the 16S rRNA, atpA, pyrH, recA, rpoA 197

and rpoD gene sequences obtained for the four clam strains are listed in Supplementary 198

Table S2. 199

The type strain C16.17T showed levels of DNA relatedness of 53% (reciprocal 45%) 200

with V. tapetis CECT 4600T, 41% (38%) with V. pomeroyi LMG 20537

T, 41% (44%) 201

with V. crassostreae CAIM 1405T, and 35% (39%) with V. anguillarum ATCC 19264

T. 202

All these wDDH values were below 70%, threshold to delimit species. On the other 203

hand, the four clam isolates showed DDH values of at least 80% (data not shown). 204

These results demonstrated that the four clam strains represent a novel species within 205

the genus Vibrio. 206

The isolates from cultured clam can be differentiated from the phylogenetically related 207

species of the genus Vibrio, by several phenotypic features (Table 1). Strains can be 208

distinguished from V. tapetis by their ability in the arginine dyhydrolsis, the 209

fermentation of glycerol and D–maltose, and the lack of the fermentation of D–mannitol 210

and amygdalin. At the same time, the clam isolates can be differentiated from V. 211

pomeroyi by their ability in fermentation of glycerol and inability in the fermentation of 212

D–mannitol. These strains can be differentiated from V. crassostreae by their inability 213

to grow at 6% of NaCl, the fermentation of D–maltose and inability in the fermentation 214

of D–mannitol and amygdalin. In addition, the analyses of FAMEs of the type strain 215

C16.17T

showed its distinct FA profile (Table S3). 216

217

The analysis of the polyphasic study clearly indicated that strains of the cluster 68 218

represent a new taxon within the genus Vibrio. The name Vibrio cortegadensis sp. nov. 219

is proposed for this new species. 220

221

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222

Description of Vibrio cortegadensis sp. nov. 223

Vibrio cortegadensis [cor.te.ga.den´.sis. N.L. gen. n. cortegadensis intended to mean 224

that the type strain was isolated from Cortegada Island in Carril (Galicia, Spain)] 225

226

Gram-negative motile rods with facultative anaerobic metabolism. All strains are 227

sensitive to the vibriostatic agent O/129, positive for arginine dihydrolase (Moeller’s 228

medium), indole reaction, production of oxidase, lipase and amylase, and for the 229

reduction of nitrates to nitrites. They are negative for the decarboxylation of lysine and 230

ornithine, Voges–Proskauer reaction, utilization of citrate, and the hydrolysis of 231

aesculin and urea. Variable reaction was observed for the catalase test, showing only 232

CMJ 9.12 strain a positive reaction. Strain Rd 13.7 is positive for ONPG test. The 233

ability to grow on TCBS is variable, being strains C16.17T and CMJ 9.12 able to grow 234

on this medium as green colonies (sucrose negative). The four isolates showed growth 235

from 1 to 3% NaCl, but not in the absence of NaCl or at salinities higher than 6% NaCl. 236

The strains are able to grow from 4 to 30ºC, but not at 37 ºC and 44 ºC. 237

All strains produce fermentation of D–glucose, D–fructose, N–acetylglucosamine, 238

aesculin, D–maltose, D–trehalose, starch, glucogen and potassium 2–ketogluconate 239

(weakly reaction), but not of erythritol, D–arabinose, L–xylose, D–adonitol, methyl–240

βD–xylopyranoside, L–sorbose, L–rhamnose, dulcytol, inositol, D–sorbitol, methyl–241

αD–mannopyranoside, methyl–αD–glucopyranoside, amygdalin, arbutin, salicin, D–242

cellobiose, D–lactose, D–melibiose, D–sucrose, inuline, D–melezitose, D–raffinose, 243

xylitol, gentiobiose, D–turanose, D–lyxose, D–tagatose, D–fucose, L–fucose, D–244

arabitol, L–arabitol, potassium gluconate and potassium 5–ketogluconate. In the API 245

ZYMsystem, all strains show positive reactions for alkaline phosphatase, esterase, 246

esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naftol–AS–247

BI–phosphohydrolase. The major fatty acids of the type strain C16.17T are C12:0 3OH 248

(4.3%), C16:0 (22.6%), summed feature in 3 (comprising C16:1 ω7c and/or C16:1 ω6c) 249

(47.9%), and summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c)(9.9%). 250

The type strain C16.17T (=CECT 7227

T= LMG 27474

T) was isolated in the north–251

western coast of Spain (Galicia), from healthy cultured clams, Venerupis decussata. 252

Isolates CMJ 9.12 (=CECT 8125= LMG 27475), CMJ 12.11 (=LMG 27427) and Rd 253

13.7(= LMG 27476) were also deposited at culture collections as reference strains of the 254

species. 255

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256

Acknowledgements 257

This work was supported in part by grant AGL–2010–18438 from the Ministerio de 258

Ciencia e Innovación (Ministry of Science and Innovation) (Spain). A. L. acknowledges 259

the Ministerio de Economía y Competitividad (Ministry of Economy and 260

Competitiveness) (Spain) for a research fellowship. 261

262

Conflict of interest 263

The authors declare that they have no conflict of interest. 264

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364

365

366

367 368

13

369 Figure legends 370

371 Figure 1. Phylogenetic position of the four isolates according to 16S rRNA gene 372

sequence analysis. The tree is a NJ tree; Vibrio cholera was used as an outgroup. 373

GeneBank sequence accession numbers are given in parentheses. Numbers at the 374

nodes show the percentage bootstrap values (only values higher than 50% are 375

shown). Bar, 0.002 substitutions per nucleotide position. 376

377

378

Figure 2. Phylogenetic position of the four isolates according to MLSA of the five 379

housekeeping genes atpA, pyrH, recA, rpoA and rpoD, and the 16S rRNA gene. The 380

tree is a NJ tree. Numbers at the nodes show the percentage bootstrap values (only 381

values higher than 50% are shown). Bar, 0.02 substitutions per nucleotide position. 382

383 384 385

Table 1. Phenotypic characteristics for distinguishing V. cortegadensis sp. nov. from related Vibrio species.

Characteristics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

ADH + – – – + + + + – + + + + + – – +

Indole + + – + + + + – + + + + + + + + +

TCBS G G Y Y Y G Y Y G Y Y G G Y G Y Y

Growth with:

3% NaCl + – + + + + + + + + + + + – + + +

6% NaCl – – + + + – + + + + + + + – + + +

Fermentation of:

Glycerol + – + + + – – – + – – – – – – – –

D-galactose V (+) + + + + – – – – – – + – – – + +

D-mannose V (–) + + + + – – – + – – + – – – + +

D-maltose + + + + + – – – + – – + – – – + +

D-glucose + + + – + + – + + – + + + – + + +

D-mannitol – + + + + + + + + + + + + + + + +

Amygdalin – + – – – – + – – + – – – + + – +

All data were obtained concurrently in this study. +, positive; –, negative; V, variable (in parenthesis is indicated the result for the type strain);

G: green colonies on TCBS; Y: yellow colonies on TCBS. Taxa are indicated as: 1, V. cortegadensis (4 strains); 2, V. tapetis CECT 4600T; 3,

V. atlanticus LMG 24300T; 4, V. artabrorum LMG 23865

T; 5, V. celticus CECT 7224

T; 6, V. chagassi LMG 21353

T; 7, V. crassostreae LMG

20537T; 8, V. cyclitrophicus LMG 21359

T; 9, V. gallaecicus LMG 24045

T; 10, V. gigantis LMG 22741

T; 11, V. kanaloae LMG 20539

T; 12, V.

lentus CECT 5110T; 13, V. pomeroyi LMG 20537

T; 14, V. splendidus CECT 628

T; 15, V. tasmaniensis LMG 20012

T; 16, V. toranzoniae CECT

7225T; 17, V. anguillarum 19264

T.

Figure 1Click here to download high resolution image

Figure 2Click here to download high resolution image

Description of the new species Vibrio cortegadensis sp. nov., isolated from clams

Aide Lasa, Ana L. Diéguez, and Jesús L. Romalde *

.

Supplementary Material

Supplementary MaterialClick here to download attachment to manuscript: Supplementary material.doc

Figure S1. Phylogenetic tree based on partial 16S rRNA gene sequences, obtained by

the Maximum Likelihood method with the K2+G+I model. GeneBank sequence

accession numbers are given in parentheses. Numbers at the nodes show the percentages

bootstrap values (only values higher than 50% are shown). Bar, 0.01 substitutions per

nucleotide position.

Vibrio kanaloae LMG 20539T (AM162657)

Vibrio lentus CECT 5110T (NR 028926)

Vibrio hemicentroti DSM 26178T (JX204734)

Vibrio atlanticus LMG 24300T (EF599163)

Vibrio tasmaniensis LMG 21574T (AJ514912)

Vibrio cyclitrophicus LMG 21359T (AM162656)

Vibrio splendidus ATCC 33125T (X74724) Vibrio gallaecicus CECT 7244T (NR 044520)

Vibrio gigantis LGP 13T (AJ582810)

Vibrio celticus LMG 23850T (EF599162)

Vibrio artabrorum LMG 23865T (EF599164)

Vibrio toranzoniae CECT 7225T (HE978310)

Vibrio pomeroyi LMG 20537T (AJ491290)

Vibrio chagasii R-3712T (AJ316199)

Vibrio crassostreae CAIM 1405T (EF094887)

Vibrio tapetis CECT 4600T (HE795129)

CMJ 9.12 (HF955038)

Rd 13.7 (HF955040)

C 16.17T (HF955037)

CMJ 12.11 (HF955039)

Vibrio pacinii LMG 19999T (AJ316194)

Vibrio ordalii ATCC 33509T (AEZC01000173)

Vibrio anguillarum ATCC 19264T (X16895)

Vibrio aestuarianus ATCC 35048T (X74689)

Vibrio mediterranei LMG11258T (HM771351) Vibrio shilonii AK1T (ABCH01000080)

Vibrio scophthalmi LMG 19158T (HM771340)

Vibrio diazotrophicus LMG7893T (X56577) Vibrio plantisponsor MSSRF60T (GQ352641)

Vibrio vulnificus LMG13545T (X76333) Vibrio azureus LC2-005T (AB428897)

Vibrio sagamiensis LC2-047TT (AB428909)

Vibrio jasicida TCFB 0772T (AB562589)

Vibrio owensii DY05T (GU018180)

Vibrio corallilyticus LMG 20984T (AJ440005)

Vibrio nereis ATCC 25917T (HQ890463) Vibrio harveyi LMG4044T (AY750575)

Vibrio variabilis R-40492T (GU929924)

Vibrio maritimus R-40493T (GU929925)

Vibrio nigripulchritudo LMG 3896T (HM771352) Vibrio halioticoli LMG 18542T (AB000390)

Vibrio cholerae CECT 514T (NR 044853)

62

100

87

54

80

90

99

67

55

0.01

Vibrio cortegadensis sp. nov.

Figure S2. Phylogenetic trees based on partial housekeeping gene sequences, obtained

by the neighbor joining method: a) atpA, b) pyrH, c) recA, d) rpoA, and e) rpoD genes.

Vibrio cholerae LMG 21698T was used as an out-group. Numbers at the nodes show the

percentage bootstrap values (only values higher than 50% are shown). Bar, substitutions

per nucleotide position.

A

Vibrio celticus CECT 7224T (FN582232) Vibrio cyclitrophicus LMG 21359T (EF601304)

Vibrio crassostreae LMG 22240T (EU541557) Vibrio gigantis DSM 18531T (EU541556) Vibrio gallaecicus CECT 7244T (EU541559)

Vibrio kanaloae LMG 20539T (EF601307) Vibrio atlanticus CECT 7223T (FN582252)

Vibrio tasmaniensis LMG 20012T (EF601325) Vibrio toranzoniae CECT 7225T (HE820043)

Vibrio lentus CECT 5110T (EU541558) Vibrio splendidus LMG 19031T (EF601244)

Vibrio pomeroyi LMG 20537T (EF601318) Vibrio artabrorum CECT 7226T (FN668898)

Vibrio chagasii LMG 21353T (EF601280) Rd 13.7 C 16.17T CMJ 9.12 CMJ 12.11

Vibrio tapetis CECT 4600T (HE795159)

Vibrio anguillarum LMG 4437T (EF601227)

Vibrio cholerae CECT 514T

(HE805628)

89 65

100 86

91

100

60 98 97 92

88

98

58

0.02

Vibrio cortegadensis sp. nov.

B

Vibrio cyclitrophicus LMG 21359T (EU871958) Vibrio pomeroyi LMG 20537T (EU871960)

Vibrio kanaloae LMG 20539T (FN908851) Vibrio toranzoniae CECT 7225T (HE820044)

Vibrio chagasii LMG 21353T (EU118252) Vibrio atlanticus CECT 7223T (FN582266)

Vibrio tasmaniensis LMG 20012T (EU871961) Vibrio lentus CECT 5110T (EU871959)

Vibrio crassostreae LMG 2240T (EU871948) Vibrio celticus CECT 7224T (FN582244)

Vibrio gigantis DSM 18531T (EU871951) Vibrio splendidus LMG 19031T (EU871962)

Vibrio artabrorum CECT 7226T (FN668929) Vibrio gallaecicus CECT 7244T (EU871963) CMJ 12.11

C 16.17T CMJ 9.12 Rd 13.7

Vibrio anguillarum M3 Vibrio tapetis CECT 4600T (HE795189)

Vibrio cholerae LMG 21698T (EF990257)

70 100

100

100

56

86

84

57

65

0.02

Vibrio cortegadensis sp. nov.

C

Vibrio gigantis DSM 18531T (EU541593) Vibrio pomeroyi LMG 20537T (AJ842497)

Vibrio toranzoniae CECT 7225T (HE820039) Vibrio crassostreae LMG 22240T (EU541594)

Vibrio artabrorum CECT 7226T (EU541588) Vibrio celticus CECT 7224T (EU541590) Vibrio kanaloae LMG 20539T (AJ842450)

Vibrio atlanticus CECT 7223T (EU541589) Vibrio tasmaniensis LMG 20012T (AJ842515)

Vibrio lentus LMG 21034T (AJ842452) Vibrio splendidus LMG 19031T (AJ842511)

Vibrio cyclitrophicus LMG 21359T (AJ842405) Vibrio chagasii LMG 21353T (AJ842385)

Vibrio gallaecicus CECT 7244T (EU5415879) Vibrio tapetis CECT 4600T (HE795219)

C16.17T CMJ 12.11

CMJ 9.12 Rd 13.7

Vibrio anguillarum LMG 4437T (AJ842375)

Vibrio cholerae CECT 514T (AM942078)

100

54

64

98

77 99

96 97

74

86

94

100

71

78

0.02

Vibrio cortegadensis sp. nov.

D

Vibrio cyclitrophicus LMG 21359T (AJ842592) Vibrio splendidus LMG 19031T (AJ842725)

Vibrio crassostreae LMG 22240T (EU541574) Vibrio celticus CECT 7224T (EU541570) Vibrio gigantis DSM 18531T (EU541573)

Vibrio pomeroyi LMG 20537T (AJ842684) Vibrio chagasii LMG 21353T (AJ842572)

Vibrio artabrorum CECT 7226T (EU541568) Vibrio atlanticus CECT 7223T (EU541569) Vibrio tasmaniensis LMG 20012T (AJ842731)

Vibrio kanaloae LMG 20539T (AJ842637) Vibrio toranzoniae CECT 7225T (HE820031)

Vibrio lentus LMG 21034T (AJ842639) Vibrio gallaecicus CECT 7244T (EU541566)

Rd 13.7 CMJ 9.12 C16.17T CMJ 12.11

Vibrio anguillarum LMG 4437T (AJ842561)

Vibrio cholerae CECT 514T (HE805630) Vibrio tapetis CECT 4600T (HE795340)

93 55

100

52

100

80 100

56 67

60

65

54

0.01

Vibrio cortegadensis sp. nov.

E

Vibrio chagasii LMG 21353T (AY751356) Vibrio pomeroyi LMG 20537T (AY751353)

Vibrio crassostreae LMG 22240T (AY751351) Vibrio gigantis DSM 18531T (AY751347)

Vibrio celticus CECT 7224T (FN582243) Vibrio lentus CIP 107166 (AY751358)

Vibrio tasmaniensis LMG 20012T (AY751354) Vibrio kanaloae LMG 20539T (AY751352) Vibrio atlanticus CECT 7223T (FN582265)

Vibrio splendidus LMG 4042T (AY751355) Vibrio toranzoniae CECT 7225T (HE820035)

Vibrio artabrorum CECT 7226T (FN668926) Vibrio cyclitrophicus LMG 21359T (AY751371)

Vibrio gallaecicus CECT 7244T (FN908850) C 16.17T

Rd 13.7 CMJ 9.12 CMJ 12.11

Vibrio tapetis CECT 4600T (HF542104)

Vibrio anguillarum ATCC 19624T (HE820026)

Vibrio cholerae CECT 514T (AM942060)

100

100

100 93

95

78

100 79

79 66

82

56

85

94

51 76

0.05

Vibrio cortegadensis sp. nov.

Figure S3. Phylogenetic tree on concatenated sequences of the housekeeping genes

atpA, pyrH, recA, rpoA and rpoD obtained by the Maximum Likelihood method with

the GTR+G+I model. Numbers at the nodes show the percentages bootstrap values

(only values higher than 50% are shown). Bar, 0.05 substitutions per nucleotide

position.

Vibrio atlanticus LMG 24300T

Vibrio tasmaniensis LMG 21574T

Vibrio lentus 4OM4T CECT 5110T

Vibrio splendidus ATCC 33125T Vibrio kanaloae LMG 20539T

Vibrio toranzoniae CECT 7225T

Vibrio celticus LMG 23850T Vibrio cyclitrophicus LMG 21359T

Vibrio gigantis LGP 13T Vibrio pomeroyi LMG 20537T

Vibrio crassostreae CAIM 1405T Vibrio artabrorum LMG 23865T

Vibrio gallaecicus CECT 7244T

Vibrio chagasii LMG R3712T

C 16.17T Rd 13.7

CMJ 9.12 CMJ 12.11

Vibrio tapetis CECT 4600T Vibrio anguillarum ATCC 19264T

Vibrio cholerae CECT 514T

100

100 96

99

100

100

99

54

62

72

57

81

73

51

0.05

Vibrio cortegadensis sp. nov.

Table S1. Differentiating phenotypic features for the four strains.

Test Strain

C16.17 CMJ 9.12 CMJ 12.11 Rd 13.7 Catalase – + – –

TCBS Green Green NG NG

ONPG – – – +

Fermentation of:

Glycerol + + – +

L-Arabinose – – – +

D-Xilose + – – –

D-galactose + – – –

D-mannose – – + –

D-mannitol – + + – NG: No growth detected.

Table S2. Genbank accession number of the 16S rRNA, recA, rpoA, rpoD, atpA and

pyrH gene sequences of the four strains of Vibrio cortegadensis sp. nov. investigated in

this study.

Table S3. Fatty acid composition (%) of the type strain C 16.17T. Cellular fatty acid

analysis was carried out according to the MIDI Microbial Identification System from

24h bacterial cultures on Marine agar.

Fatty acid C 16.17T V. pomeroyi V. crassostreae

Summed features*

2 4,44 ± 0,1% 0.0 2·6±0·1  %

3 47,88 ± 0,2% 32.9±1.6 39·4±1  %

8 9,93 ± 0,1% 7.6±1.8 7±1·3  %

12:0 3OH 4,30 ± 0,1% 3.9±0.6 3·3±0·2  %

14:0 2,23 ± 0,1% 10.5±0.4 5·4±1  %

16:0 22,62 ± 0,1% 29.2±1.7 17·3±1·3  %

18:0 1,15 ± 0,1% 1.6±0.2 0%

Fatty acid traits shown here correspond to those that are present with more than 1%.

* 2, 12:0 aldehyde, and/or unidentified fatty acid with equivalent chain-length value of

10.928; 3, 16:1 ω7c and/or 16:1 ω6c; 8 18:1 ω7c and/or 18:1 ω6c.

Strain 16S atpA pyrH recA rpoA rpoD

C 16.17T HF955037 HF955041 HF955045 HF955049 HF955053 HF955057

CMJ 9.12 HF955038 HF955042 HF955046 HF955050 HF955054 HF955058

CMJ 12.11 HF955039 HF955043 HF955047 HF955051 HF955055 HF955059

Rd 13.7 HF955040 HF955044 HF955048 HF955052 HF955056 HF955060