R E S E A R C H A R T I C L E

Denaturinggradientgel electrophoresis analysesoftheverticaldistributionanddiversityofVibrio spp.populations in theCariacoBasinMaria Alexandra Garcıa-Amado1, Lorelei Bozo-Hurtado2, Yrene Astor3, Paula Suarez2 &Andrei Chistoserdov4

1Centro de Biofısica y Bioquımica, Instituto Venezolano de Investigaciones Cientıficas, Caracas, Venezuela; 2Departamento de Biologıa de Organismos,

Universidad Simon Bolıvar, Caracas, Venezuela; 3EDIMAR, Fundacion de Ciencias Naturales La Salle, Margarita Island, Venezuela; and 4Department of

Biology, University of Louisiana at Lafayette, LA, USA

Correspondence: Maria Alexandra Garcıa-

Amado, Laboratorio de Fisiologıa

Gastrointestinal, Centro de Biofısica y

Bioquımica, Instituto Venezolano de

Investigaciones Cientıficas, Altos de Pipe. Edo

Miranda,Venezuela, 1020-A. Tel.: 158 212

504 1855; fax: 158 212 504 1093; e-mail:

magarcia@ivic.gob.ve

Received 11 November 2010; revised 31

January 2011; accepted 10 April 2011.

Final version published online 18 May 2011.

DOI:10.1111/j.1574-6941.2011.01116.x

Editor: Patricia Sobecky

Keywords

Vibrio, Cariaco, PCR-DGGE.

Abstract

The Cariaco system is the second largest permanently anoxic marine water body in

the world. Its water column is characterized by a pronounced vertical layering of

microbial communities. The goal of our study was to investigate the vertical

distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using

denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA

fragments. Representatives of the Vibrio genus were detected by nested and direct

PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that

only 11 different operational taxonomic units (OTU) are identified as Vibrio

species. Between one and five OTUs were detected at each depth and the most

common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with

Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the

anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius,

respectively. Several Vibrio species detected are potentially pathogenic to human,

prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio

shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to

specific depths strata, suggesting that this genus is a natural component of the

microbial communities in this marine redox environment.

Introduction

The genus Vibrio from the family Vibrionaceae belongs to the

class Gammaproteobacteria and contain 4 63 recognized

species of gram-negative rods, which are widely distributed

in the estuarine and marine environments (Thompson et al.,

2004). In general, Vibrio spp. tolerate a wide range of

salinities and tend to be more common in warm waters,

notably when temperatures exceed 17 1C (Eiler et al., 2006).

There are indications that vibrios play a role in nutrient

cycling in aquatic environments by taking up dissolved

organic matter and they may provide essential polyunsatu-

rated fatty acids to the aquatic food web, which many

aquatic organisms cannot produce de novo (Thompson

et al., 2004).

Most existing studies of the Vibrio ecology have been

based on traditional culture-dependent techniques; how-

ever, these are slow, laborious and often require several days

to be performed (Thompson et al., 2004; Igbinosa &

Okoh, 2008). Moreover, vibrios can enter a viable, but not

culturable state, where the bacteria are still alive, but fail

to grow on conventional bacteriological media (Oliver,

2010). For these reasons, the application of molecular

cultivation-independent techniques, like denaturing gradi-

ent gel electrophoresis of PCR-amplified DNA fragments

(PCR-DGGE), has provided efficient means of detecting and

identifying marine bacteria, including vibrios (Eiler &

Bertilsson, 2006).

Several investigations have demonstrated that Vibriona-

ceae can be isolated from the water column, sediments and

intestinal tracts of invertebrates from deep-sea environ-

ments (Delong & Yayanos, 1986; Raguenes et al., 1997;

Reen et al., 2006). The Cariaco Basin offers a particularly

interesting deep-sea system for a study, because it is the

FEMS Microbiol Ecol 77 (2011) 347–356 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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second largest permanently anoxic marine water body

in the world. The Cariaco Basin is located on the northern

continental shelf of Venezuela in the Caribbean Sea,

where depending on a year the oxygen is absent below

depths from 220 to 400 m (Madrid et al., 2001; Stoeck

et al., 2003; Tedesco et al., 2007). The basin water column is

characterized by a pronounced and predictable vertical

stratification of microbial communities and composed of

three layers: (1) oxic with the most complex trophic

structure in carbon metabolism; (2) redox cline, biogeo-

chemically dominated by chemolithoautotrophy; and

(3) anoxic zone that appears to support processes like

fermentation, sulfate reduction, methanogenesis and

anaerobic methane oxidation (Taylor et al., 2003). Accord-

ingly, microbial communities associated with such

biogeochemical zonation change with the depth in the

Cariaco Basin water column (Madrid et al., 2001;

Lin et al., 2008). Previous studies have reported the presence

of Gammaproteobacteria throughout the water column

of the basin (Lin et al., 2006). However, the presence and

diversity Vibrio populations have not been investigated so

far. Culturing on various media revealed the presence of

only one strain of Vibrio alginolyticus throughout the

entire depth profile (i.e. from the oxic to the anoxic zone;

A. Chistoserdov, unpublished data). Because molecular-

based techniques provide a more comprehensive picture of

the microbial diversity, in the present study, we chose to

analyze the vertical distribution and diversity of Vibrio spp.

in the stratified water column of the Cariaco Basin by PCR-

DGGE.

Materials and methods

Sampling site and physicochemicalmeasurements

The Cariaco Basin is currently the focus of the CArbon

Retention In A Colored Ocean (CARIACO) time series

program, a cooperative United States–Venezuelan research

project (http://www.imars.usf.edu/CAR/) (Muller-Karger

et al., 2001). Located on the Venezuelan continental shelf

(Fig. 1), the Cariaco Basin is 160-km long, 70-km wide and

has a maximum depth of about 1400 m. It is divided into

two sub-basins by a saddle at about 900 m. The basin is

isolated from the rest of the Caribbean by a 150 m depth sill

connecting Isla Margarita to Cabo Codera on the Venezue-

lan mainland (Muller-Karger et al., 2000).

Water column sampling was conducted aboard the R/V

Hermano Gines, operated by Estacion de Investigaciones

Marinas (EDIMAR), Fundacion la Salle de Ciencias Natur-

ales, Margarita Island, Venezuela. Water samples were col-

lected using a SeaBird rosette accommodating 12 TFE-lined,

12-L Niskin bottles. For hydrographic profiling, the rosette

was equipped with a SeaBird CTD and a YSI oxygen probe.

Two liters of seawater were collected at 10 different depths

(40, 100, 200, 230, 265, 280, 400, 500, 900, 1300 m) in the

July 8, 2008, time series cruise (CAR-147) from station A

(101300N, 641400W) situated in the eastern sub-basin of the

Cariaco system (Fig. 1). These seawater samples were filtered

through GV Durapores membrane filters (diameter

47 mm; pore size 0.2mm; Millipore, Billerica, MA); the

membrane filters were immediately immersed in 4.5 mL of

Fig. 1. Map of the Cariaco Basin sampling site.

Reproduced and modified from Astor et al. (2003).

FEMS Microbiol Ecol 77 (2011) 347–356c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

348 M.A. Garcıa-Amado et al.

autoclaved DNA extraction buffer (20 mM Tris-HCl, pH 7.8,

50 mM EDTA, 20 mM NaCl) and stored frozen (� 20 1C)

until DNA was extracted.

DNA extraction

DNA from seawater samples was extracted using two DNA

isolation protocols: the QIAamp DNA Mini Kit (Qiagen,

Valencia, CA) and a phenol: chloroform method described

by Madrid et al. (2001). For QIAamp DNA, one quarter of

each filter was sterilely cut and resuspended in 180mL of the

first buffer of the QIAamp DNA Mini Kit (according to the

manufacturer’s recommendations). Additionally, 1 mL of

supernatant (i.e. DNA extraction buffers) was centrifuged

at 14 000 g for 1 min. The supernatant generated by centri-

fugation was discarded, and DNA was purified from the

pellet also using the QIAamp DNA Mini Kit. The rest of the

filters (3/4) were cut and, along with the remaining 3.5 mL

of extraction buffer, were used to extract the DNA using the

phenol : chloroform method (Madrid et al., 2001).

Vibrio PCR

Vibrio DNA was amplified using either direct PCR or nested

PCR assays. For the direct PCR, Vibrio genus-specific primer

designed by Liu et al. (2006) (V744R 50-CAT CTG AGT GTC

AGT RTC TG-30) in combination with the bacterial primer

with a GC clamp designed by Muyzer et al. (1993) (341F-

GC: 50-CGC CCG CCG CGC GCG GCG GGC GGG GCG

GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-30)

were used. The reaction mixture contained 6mL of DNA

(approximately �50–100 ng) and 0.5 mM of each primer

(V744R and 341F-GC), 25 mL of GoTaqs Green Master Mix

reactions (Promega, Madison, WI) and water added to a

final volume of 50mL. PCR amplification was performed in a

thermal cycler (PxE Thermal Cycler, Thermo Hybaid)

according to the following program: 94 1C for 1 min, one

cycle at 94 1C for 1 min, 65 1C for 1 min, 72 1C for 3 min, 20

cycles at 94 1C for 1 min, 64 1C for 1 min, 72 1C for 3 min, 15

cycles at 94 1C for 1 min, 55 1C for 1 min and 72 1C for

3 min, followed by 72 1C for 7 min.

For nested PCR, Vibrio DNA was amplified using two sets

of primers. The first set included universal primers for the

eubacterial 16S rRNA gene described by Weisburg et al.

(1991), with modifications (AC18 50-AGA GTT TGA TCH

TGG CTY AG-30 and AC22 50-ACG GNT ACC TTG TTA

CGA CTT-30). The V744R and 341F-CG primers were used

for the second PCR round. The reaction mixture for the first

step contained 2mL of DNA (�15–30 ng of total community

DNA) and 0.5mM of each primer (AC18 and AC22), added

to a final volume of 25mL of 12.5mL of GoTaqs Green

Master Mix reactions and nuclease-free distilled water

(Promega). PCR amplification was performed in a thermal

cycler (PxE Thermal Cycler, Thermo Hybaid) according to

the following program: 95 1C for 5 min, 25 cycles at 94 1C for

30 s, 55 1C for 30 s and 72 1C for 45 s, followed by an

extension step at 72 1C for 7 min. Three microliters of the

PCR amplicons from the first step was transferred into a

second reaction mixture (50mL) and reamplified with

0.5 mM of each primer (341F-GC and 744R) under the same

PCR conditions. The negative control in both rounds of

PCR had no template in a reaction. We added a second

negative control, which is to use the negative control of the

first round of PCR as a template for the second round of

PCR. This allows the detection of minor contaminations,

arising from the first round of PCR that would otherwise

remain unnoticed. The positive control for direct and nested

PCR was prepared using 1 mL of V. alginolyticus (strain V80)

DNA (100 ng) under the reaction conditions described

above. This bacterial strain is indigenous to and was isolated

from the Cariaco Basin. The PCR products were visualized

by running an aliquot of the reaction mixture in TBE

agarose gels (1.0%), staining the gels with ethidium bromide

(0.2mg mL�1), and observing and photographing them

under UV light.

DGGE

DGGE analysis of Vibrio amplicons (50mL – entire volume

of a PCR reaction) was performed in 6% polyacrylamide

(37,5 : 1 acrylamide/bis-acrylamide) gels that contained a

40–80% linear gradient of a denaturing solution of urea plus

formamide [100% denaturing solution contains 7 M urea

and 40% (v/v) formamide]. Electrophoresis was performed

in 0.5�TAE at 60 V and 60 1C for 15 h. in a DGGE-1001-

110 System (C.B.S. Scientific Company Inc.). Gels were

stained with ethidium bromide (0.2mg mL�1) for 20 min

and visualized using a FOTO/Analyst Investigator/FX Sys-

tems (Fotodyne Incorporated, Hartland, WI).

16S rDNA sequence analysis

Separated DNA fragments were cut from DGGE gels with a

scalpel and transferred to microcentrifuge tubes. The tubes

with gel strips were placed in a freezer at � 80 1C for 2 h and

then supplemented with 0.2 g of sterile zirconia/silica beads

(BioSpec Products, Bartlesville, OK) and 500 mL of sterile

HPLC water (Fisher HealthCare). Samples were beaten in a

Mini-Beadbeater 8 for 3 min (BioCold Scientific, Fenton,

MO) and left for extraction at 4 1C overnight. A 3 mL aliquot

was used as a template for the PCR amplification of Vibrio

16S rDNA using the primers 341F (same as 341F-GC, but

without a GC clamp) and V744R and the same amplification

conditions as for the second step of nested Vibrio PCR

described above. Reamplified PCR products were purified

using a Wizards SV gel and PCR clean-up system kit

(Promega). Sequencing of one DNA strand was performed

using a BigDyes Terminator v3.1 sequencing kit according

FEMS Microbiol Ecol 77 (2011) 347–356 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

349Vertical diversity of vibrios in the Cariaco Basin

to the recommendation of the manufacturer (Applied

Biosystems, Foster City, CA). Sequencing reactions were

analyzed in a 3100 ABI DNA sequencer. Sequence quality

was verified using the CHROMAS LITE software (http://www.

technelysium.com.au/chromas_lite.html).

Phylogenetic analysis

Partial 16S rRNA gene sequences were initially compared

with sequences in the GenBank database using BLASTN

(Altschul et al., 1997) to determine their approximate

phylogenetic affiliations and then, along with the closest

GenBank matches, were aligned using the NAST Alignment

utility of the Green Genes web site (DeSantis et al., 2006b).

The sequences were checked for possible chimeras using the

CHIMERA_CHECK program at the Green Genes website (De-

Santis et al., 2006a). The sequences obtained from 67 DGGE

bands were grouped into operational taxonomic units

(OTUs) based on a 99% rRNA gene sequence similarity

level. This grouping was achieved by first performing all

possible pairwise sequence alignments using BLASTN 2.2.1

(bl2seq) (Altschul et al., 1997) at default settings and

calculating percent sequences identities, followed by cluster-

ing the sequences into OTUs. Representative sequences from

each OTU were aligned using the NAST Alignment utility

(DeSantis et al., 2006b), and a phylogenetic tree was

constructed using 350-bp-long aligned sequences from 12

OTUs using the neighbor-joining algorithm (the Jules–Can-

tor Model) provided in MOLECULAR EVOLUTIONARY GENETICS

ANALYSIS 2.1 software (MEGA, version 4) (Tamura et al., 2007).

Bootstrapping was used to estimate the reliability of phylo-

genetic reconstructions (1000 replicates). The representative

sequences determined in this study were submitted to the

GenBank database with the accession numbers

HQ456351–HQ456362.

Statistical analysis

The relation between Vibrio DGGE bands and Cariaco

depths was analyzed using cluster analysis based on Eucli-

dean distances calculated among observations for untrans-

formed data. Statistical analysis was performed using the

PAST program (http://folk.uio.no/ohammer/past/).

Results

The depth profiles of the temperature, salinity and dissolved

oxygen of the water column determined during sampling are

shown in Table 1. The temperature in seawater samples

ranged from 17.02 to 22.25 1C and the salinity values ranged

between 26.25 and 36.90 PSU. The dissolved O2 concentra-

tion peaked at 40 m (4.7 mg L�1) and then declined drasti-

cally at depths below 200 m. Thus, the analyses of all samples

allowed us to study the diversity of Vibrio spp. in the oxic,

redox cline and anoxic zones.

16S rDNA of bacteria belonging to genus Vibrio was

detected by direct PCR only in three samples. The first of

these samples was collected from 40 m and total DNA was

extracted using a phenol/chloroform extraction method.

The second and third samples were collected from 40 and

100 m and total DNA was extracted from a filter using a

QIAamp DNA Mini Kit. The application of nested PCR

produced Vibrio 16S rDNA amplicons for all samples. All

positive Vibrio 16S rDNA amplicons (generated by both

direct and nested PCR) were analyzed in DGGE gels (Fig. 2).

The banding patterns in the gels were similar regardless of

the DNA extraction method used. We observed that 16S

rDNA amplicons generated by nested-PCR produced more

bands than those produced by direct PCR.

The Cariaco water column at the time of sampling can be

divided into three zones as follows: oxic (0–200 m), redox

cline (230–280 m) and anoxic (400–1300 m) zone. Clear

differences in the band patterns of the DGGE gel were

observed along the depth profile (Fig. 2). The oxic zone

samples contained fewer Vibrio bands than the redox inter-

face samples, but Vibrio spp. were detected by direct PCR only

in the oxic zone. Anoxic zone samples (400, 500 and 900 m)

show band patterns similar to those of the redox cline zone.

The band pattern of the deepest sample (1300 m) was unique

regardless of the DNA extraction method used, suggesting

that the Vibrio community uniqueness at this depth is not an

artifact of sample handling or a DNA isolation method.

Sixty-seven DGGE bands were excised, sequenced and

their sequences compared with each other and the existing

sequences in the NCBI public database by the BLAST algo-

rithm. Then, the DGGE band sequences were grouped into

OTUs based on 99% similarity to each other and were

tentatively assigned to a described Vibrio species. A pairwise

comparison of the DGGE band sequences showed that the

67 sequenced DGGE bands (including those for the positive

control) in reality fall only into 12 different OTUs (shown in

Table 1. Physicochemical parameters in the water column of the

Cariaco Basin in July 2008

Seawater sample

(m)

Temperature

( 1C)

Salinity

(PSU)

Dissolved O2

(mg L�1)

40 22.25 36.90 4.7006

100 20.28 36.71 4.6257

200 18.03 36.41 0.0465

230 17.91 36.40 0.0176

265 17.81 36.38 0.0159

280 17.74 36.37 0.0154

400 17.43 36.33 0.0136

500 17.25 36.30 0.0128

900 17.06 36.26 0.0113

1300 17.02 36.25 0.0109

FEMS Microbiol Ecol 77 (2011) 347–356c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

350 M.A. Garcıa-Amado et al.

Fig. 3). This is not unusual; one microorganism can produce

a series of distinct bands in a DGGE gel and these bands may

have identical or near identical sequences (e.g., see lane ‘C1’

in Fig. 2). The majority of the sequences were grouped into

five OTUs. OTU 1 with 23 sequences (bands 1, 2, 3, 6, 7, 8,

14, 15, 20, 22, 23, 37, 39, 41, 59, 60, 81, 82, 83, 85, 86, 102

and 286) formed a cluster with Vibrio splendidus in the

phylogenetic tree in Fig. 3. OTU 2 with 15 sequences (bands

19, 21, 29, 33, 42, 43, 47, 48, 50, 103, 104, 115, 128, 235 and

267) clustered with Vibrio fortis. OTU 3 with 7 sequences

(bands 36, 38, 57, 58, 107, 109 and 268) clustered with

Vibrio orientalis. OTU 4 with 3 sequences (bands 49, 116 and

236) clustered with Vibrio neptunius and OTU 10 and OTU

26 clustered along with Vibrio fischeri. Additionally, five

DGGE bands with numbers 11, 12, 84, 101 and 285 were

unique sequences with o 99% similarities to other DGGE

bands or each other. These sequences were assigned to OTU

11, OTU 12, OTU 84, OTU 101 and OTU 285. OTU 101

clustered in the phylogentic tree with OTU 10 and OTU 26

and more distantly with V. fischeri. OTUs 11 and 12

clustered with Vibrio shilonii, OTU 84 clustered with Vibrio

parahaemolyticus and OTU 285 clustered with the OTU 2

and, hence, more distantly with V. fortis. The positive

control DNA (i.e. a pure culture of V. alginolyticus isolated

from the Cariaco Basin) generated a banding pattern

different from those for the Cariaco samples. All sequenced

bands from positive control lanes (i.e. bands 67, 68, 69, 70,

71, 72, 73, 74, 75, 77, 79 and 80) had a 99% rRNA gene

sequence similarity, with V. alginolyticus forming their own

group called ‘OTU control’ (Figs 2 and 3).

Thus, between one and five OTUs were found for each

depth. The most common OTUs were OTU 1 and OTU 2.

Three OTUs (OTU 11, OTU 12 and OTU 84) were present

only in the oxic zone, one OTU (OTU 285) was present only

in the redox cline zone and three OTUs (OTU 3, OTU 4 and

OTU 101) were present only in the anoxic zone.

To compare the Vibrio distribution throughout at depths

of the Cariaco water column, a dendrogram (Fig. 4) was

generated using the DGGE band patterns (positive control

removed). The overall Vibrio community from different

depths falls into two clusters. The patterns of the two upper

depths (40 and 100 m) clustered together and were sepa-

rated from other depth patterns, which also clustered

together. It was interesting to note that the oxygen values

are the highest at 40 and 100 m (Table 1); it becomes 100-

fold more depleted at 200 m depth. This suggests that

oxygen is one of the major environmental parameters

shaping Vibrio community composition.

Discussion

The environmental patterns observed during our sampling

are in agreement with those reported for the Cariaco Basin

by the CARICO time series (http://www.imars.usf.edu/

CAR/). The oxygen concentration was 4.66 mg L�1 in the

photic zone and decreased to 0.0465 mg L�1 at 200 m to

0.0109 mg L�1 at the bottom. Thus, during sampling, the

physicochemical parameters that could possibly shape

Vibrio communities were typical for the Cariaco Basin and

Fig. 2. Separation of direct and nested PCR

products amplified with Vibrio genus-specific 16S

rDNA primers in 6% polyacrylamide gels with a

40–80% denaturing gradient (increasing

gradient of a denaturant from top to bottom).

(a) Vibrio spp. 16S rDNA amplified from DNA

isolated using the phenol/chloroform method.

(b) Vibrio spp. 16S rDNA amplified from DNA

isolated using the Qiagen method. A number

above each lane indicates the depth in meters

from which a sample was collected. All 16S rDNA

amplicons except those in red-colored boxes are

generated by nested PCR; those in the

red-colored box are generated by direct PCR.

Lanes marked with ‘C1’ contain amplified 16S

rDNA from the positive control (Vibrio

alginolyticus). Lanes marked ‘C� ’ contain

negative controls: no DNA introduced in PCR

reactions. The bands cut and sequenced were

identified with a number on their left side. The

bands numbers 235, 236, 267, 268, 285 and 286

are not shown in this figure.

FEMS Microbiol Ecol 77 (2011) 347–356 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

351Vertical diversity of vibrios in the Cariaco Basin

similar to those reported by other authors (Scranton et al.,

1987; Percy et al., 2008).

Two different PCR methods were applied to analyze the

Vibrio genus diversity in the water column of the Cariaco

Basin. Using the first approach – direct PCR – the Vibrio

genus was detected only in three samples from the oxic zone

out of a total of 30 samples, producing a few bands in the

DGGE gels. These patterns with few bands likely represent

the dominant Vibrio sp. well adapted and thriving in an

oxygen-rich environment. The nested PCR, however,

yielded Vibrio 16S rDNA products for all samples, with

additional bands observed in DGGE patterns for the 40 and

100 m samples. This was expected, because nested PCR as a

method has a much higher sensitivity than direct PCR

allowing amplification of sequences present in extremely

low numbers.

Sequence analyses of 67 DGGE bands revealed that a

single Vibrio species may generate multiple bands in DGGE

gels. This can serve either as a reflection of a tremendous

genomic variation among a collection of co-occurring

Vibrio strains or may just be an artifact of DGGE. The

presence of multiple bands did not place any constrains on

Fig. 3. Phylogenetic tree of partial 16S rRNA

gene sequences belonging to Vibrio spp. from

the Cariaco Basin. Tree was constructed using

the neighbor-joining algorithm. Bootstrap values

are based on 1000 replicates each and no values

are given for groups with bootstrap values

o 50%. The scale bar represents a 0.01 (1%)

nucleotide sequence difference. The number in

parenthesis following an OTU name represents

the numbers of sequences assigned to each OTU.

FEMS Microbiol Ecol 77 (2011) 347–356c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

352 M.A. Garcıa-Amado et al.

our analyses of Vibrio spp. populations, perhaps, because we

managed to sequence at least one representative of the

majority (if not all) of the bands in DGGE gels. Studies by

other authors (Le Roux et al., 2004; Thompson et al., 2005;

Tian et al., 2008; Chimetto et al., 2009) have suggested that

exclusive comparisons of 16S rRNA genes may provide a

limited resolution for identifying Vibrio spp. For example,

Tian and collaborators (2008) suggest that 16S rRNA gene is

suitable to describe the relationships among bacteria with a

larger genetic distance, whereas the gyrB gene is more

appropriate for identifying intergeneric relationships. This

clearly was not the case in the present study.

Our work demonstrates that Vibrio spp. populations are

widely distributed throughout the sampled depth profile

(40–1300 m), suggesting that bacteria belonging to this

genus are a natural component of the microbial commu-

nities in the water column of the Cariaco Basin. Moreover,

the Vibrio distribution in the Cariaco water column was not

random. In the oxic zone (0–200 m), we detected six OTUs

(OTU 1, OTU 2, OTU 10, OTU 11, OTU 12 and OTU 84)

corresponding to different Vibrio spp., of which OTU 1,

represented by two major and a few minor DGGE bands,

was the most prominent. OTU 1 was the only Vibrio OTU

amplified by directed PCR, suggesting that this Vibrio sp. is

not only the most widely distributed but also the most

abundant one in an upper portion (40 and 100 m) of the

oxic zone. It is known that vibrios utilize the dissolved

organic matter (Thompson et al., 2004) and the Cariaco

Basin seasonally has high levels of primary production

stimulated by a large supply of nutrients from the upwelled

Atlantic subtropical underwater (Richards, 1975; Muller-

Karger et al., 2001). Therefore, it is reasonable to speculate

that the oxic habitat is native for OTU 1 vibrios, where they

are present in relatively large numbers and feed on organic

matter. Their presence in lower numbers at all other depths

may then be explained by their association with sinking

marine snow particles. The dendrogram in Fig. 4 confirms

the similarity of Vibrio community at 40 and 100 m depths

to each other, whereas the Vibrio community from the depth

of 200 m was more similar to those from the redox cline and

anoxic zones. Vibrios are capable of both respiratory and

fermentative metabolism and use oxygen as an electron

acceptor for respiration (Igbinosa & Okoh, 2008). Perhaps

100-fold oxygen depletion affects the presence and decreases

the abundance of most Vibrio spp., because they would have

to switch from more a metabolically efficient respiration

mode to less metabolically efficient fermentation. Thus, it

may explain why we observed Vibrio spp. below 100 m only

by nested PCR.

Three OTUs (OTU 11, OTU 12 and OTU 84) were found

only in the oxic zone. OTUs 11 and 12 were clustered with

V. shilonii and the OTU 84 was clustered with V. parahae-

molyticus (Fig. 3). Vibrio shilonii (also known as Vibrio shiloi

and Vibrio mediterranei) can be associated with healthy,

bleached or necrotic corals in Caribbean, Mediterranean and

Pacific reefs (Banin et al., 2000; Kushmaro et al., 2001;

Thompson et al., 2001, 2004; Chimetto et al., 2009; Teplitski

& Ritchie, 2009). Vibrio parahaemolyticus, a frequent food-

borne human pathogen, is a normal inhabitant of estuarine

and marine environments, and is frequently isolated from

seawater and seafood (Thompson et al., 2004; Igbinosa &

Okoh, 2008). In Venezuela, V. parahaemolyticus was found

in bivalves (Arca zebra and Perna perna) from an area near

the Cariaco Basin (Grau et al., 2004; Munoz et al., 2008), but

the presence of V. shilonii in Venezuela coastal waters has not

so far been reported.

In the redox cline zone (230–280 m), we detected four

OTUs (ubiquitous OTU 1, OTU 2, OTU 26 and OTU 285),

of which only OTU 285 was found exclusively in the redox

cline zone. The OTU 285 had somewhat lower 16S rDNA

sequence similarity (around 97%) to known species (Fig. 3)

and had the OTU 2 group as its closest relative.

Five OTUs (OTU 1, OTU 2, OTU 3, OTU 4 and OTU

101) were detected in the anoxic zone (400–1300 m). How-

ever, only OTU 3 and OTU 4 were exclusive to this

environment and were prominent only at 1300 and 900 m,

respectively. The OTU 3 was clustered with V. orientalis and

the OTU 4 was clustered with V. neptunius. Vibrio neptunius

has originally been isolated from larvae of the bivalve

Nodipecten nodosus (Thompson et al., 2003a). Additionally,

V. neptunius has been reported as a pathogen for rainbow

trout (Oncorhynchus mykiss) and Artemia nauplii (Austin

Fig. 4. Dendrogram showing the relationship between Vibrio spp.

communities throughout at depths of the Cariaco water column,

calculated on the basis of the Euclidean distances, using the DGGE band

patterns.

FEMS Microbiol Ecol 77 (2011) 347–356 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

353Vertical diversity of vibrios in the Cariaco Basin

et al., 2005). Vibrio orientalis has been isolated from seawater

of the coast of China (Yang et al., 1983) and from mussels

(Mytilus galloprovincialis) from the Mar Piccolo of Taranto

(Italy) (Cavallo & Stabili, 2002). Before this work, V.

orientalis and V. neptunius have been isolated from aquatic

animals; however, these Vibrio species have not so far been

detected in deep anoxic seawater.

OTU 1 and OTU 2 were found in all zones of Cariaco

water column, but as mentioned above, the OTU 1 was most

prominent at depths with high oxygen levels. OTU 1 formed

a cluster with V. splendidus and Vibrio chagasii (Thompson

et al., 2003c), but appears to be more closely related to V.

splendidus. Le Roux et al. (2004) claim that the biochemical

tests currently available and partial 16S rDNA gene se-

quences are not sufficient to discriminate between strains

of V. splendidus and V. chagasii species. Vibrio splendidus

strains have been associated with the mortality of different

marine invertebrates (Nicolas et al., 1996; Gatesoupe et al.,

1999; Lacoste et al., 2001) and were also isolated from anoxic

intertidal sediments of the German North Sea coast (Freese

et al., 2009). This work is the first report of V. splendidus-

related sequences in anoxic tropical marine waters.

OTU 2 (15 sequences) and closely related OTU 285

clustered with V. fortis. They were detected from 200 to

1300 m depths; however, they are clearly less prominent at

1300 m. It is worth noting that the OTU 2 was barely

detectable by nested PCR in the oxic zone, but it becomes a

prominent Vibrio sp. in the redox cline and anoxic zone.

Originally, V. fortis has been isolated from white shrimp

Litopenaeus vannamei in Ecuador (Thompson et al., 2003b),

and more recently, from Brazilian coral Mussismilia hispida

(Chimetto et al., 2009). Vibrio fortis has also been reported

as a pathogen for corals (Thompson et al., 2004), rainbow

trout (O. mykiss) and A. nauplii (Austin et al., 2005). This

species had not been reported in deep seawater; however,

Raina et al. (2009) demonstrated that V. fortis and V. fischeri

can degrade sulfur compounds into acrylic acid, suggesting

a role for these species in the biogeochemical cycling of

sulfur at the Cariaco Basin.

The OTUs 10 (depth 100 m), 26 (depth 265 m) and 101

(depth 1300 m) were clustered with V. fischeri and this

clustering was supported by a bootstrap value of 87 (Fig.

3). Vibrio fischeri is best known as the specific symbiont in

light-emitting organs of certain squids and fishes, where it

produces luminescence by expressing the lux operon (Ruby,

1996; Thompson et al., 2004).

In conclusion, the fact that different Vibrio spp. were

found in the stratified water column and they show an

adhesion to a specific environment in the Cariaco water

column (OTU 1 – 40 and 100 m; OTU 2 – 230 through

900 m, OTU 3 – 1300 m, OTU 4 – 900 m) suggests that this

genus is a natural component of microbial communities in

the Cariaco Basin. It is also interesting that molecular

techniques fail to detect the only Vibrio sp., i.e. V. alginoly-

ticus, that can be cultured from the Cariaco Basin and, vice

versa, culturing failed to detect any of the several vibrios

detected by PCR-DGGE. This study revealed that many of

the Vibrio spp. detected in the Basin had close relatives able

to cause disease in aquatic animals. The most parsimonious

explanation for this is that pathogenic Vibrio spp. are

studied more intensely due to their practical importance

for humans. Further research may likely demonstrate that

these pathogens can exist as benign members of marine

microbial communities. The present study is the first to

report the detection of an environmental and opportunistic

bacterial genus such as Vibrio associated with an anoxic

basin. However, further work is required to better under-

stand the contribution of Vibrio spp. to the biological and

biogeochemical process in the Cariaco Basin waters.

Acknowledgements

The authors gratefully acknowledge Maria Jose Rodriguez

for her assistance with the DGGE training and Andres Sajo

for assistance with the statistical analysis. The authors would

like especially thank the captain and crew of the Hermano

Gines and Javier Camparo and Jose Jesus Narvaez at

Fundacion La Salle in Isla Margarita for their help and

logistical support. This work was funded by the ASM

International Fellowship, a grant from the Instituto Venezo-

lano de Investigaciones Cientıficas (IVIC) to M.A.G.-A., a

grant from the Decanato de Investigacion y Desarrollo of the

Universidad Simon Bolıvar to P.S. and the NSF grant MCB-

0348045 to A.Y.C. The hydrographic and other oceano-

graphic observations at the CARIACO Ocean Time Series

are supported by NSF Grant OCE-0326268 to Frank Muller-

Karger.

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