Download Diversity and distribution of pigmented heterotrophic bacteria in

Diversity and distribution of pigmented heterotrophic bacteria in
marine environments
Hailian Du, Nianzhi Jiao, Yaohua Hu & Yonghui Zeng
State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, China
Correspondence: Nianzhi Jiao, State Key
Laboratory of Marine Environmental Science,
Xiamen University, Xiamen, 361005, China.
Tel.: 86-592-2187869; fax: 86-592-2187869;
e-mail: [email protected]
Received 21 February 2005; revised 18
November 2005; accepted 23 November 2005.
First published online 8 February 2006.
doi:10.1111/j.1574-6941.2006.00090.x
Editor: Riks Laanbroek
Keywords
carotenoids; genetic diversity; pigmented
bacteria.
Abstract
A systematic investigation of marine pigmented heterotrophic bacteria (PHB)
based on the cultivation method and sequencing analysis of 16S rRNA genes was
conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the
abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients
from coastal to oceanic waters, with the highest values of 9.9 103 cell mL1 and
39.6%, respectively, in the Yangtze River Estuary. In contrast to the total
heterotrophic bacteria (TB) and CFU, which were present in the whole water
column, PHB were primarily confined to the euphotic zone, with the highest
abundance of PHB and ratio of PHB to CFU occurring in surface water. In total,
247 pigmented isolates were obtained during this study, and the phylogenetic
analysis showed a wide genetic diversity covering 25 genera of six phylogenetic
classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria
and Sphingobacteria were obtained mainly from the South China Sea and East
China Sea; PHB from the Pacific Ocean water were predominantly affiliated with
Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into
the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g.
golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range
between 450 and 550 nm. In conclusion, our work has revealed that PHB with
broad genetic diversity are widely distributed in the marine environment, and may
account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total
microbial community. This value might even be underestimated because it is
probable that not all pigmented bacteria were isolated. Their abundance and
genetic distribution are heavily influenced by environmental properties, such as
light and nutrition, suggesting that they have important roles in the marine
ecosystem, especially in the absorption of visible light.
Introduction
Heterotrophic bacteria play a significant role in the biogeochemical cycle of carbon and other materials such as
nitrogen and sulphur in the ocean because of their high
abundance and ubiquity (Fuhrman et al., 1989; Copley,
2002; Karl, 2002). Little is known, however, about the
heterotrophic bacteria as an important component in the
absorption of light in marine environments (Morel & Ahn,
1990; Stramski & Kiefer, 1991). Bacterial light absorption,
especially of the visible spectra in the sea, has been largely
ignored for a long time as a result of the general presumption that marine heterotrophic bacteria do not contain
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pigments with significant absorption in the visible spectral
range (Morel & Ahn, 1990; Stramski & Kiefer, 1991,1998). In
fact, a large number of heterotrophic bacteria can synthesize
carotenoids, and carotenoid-rich species have been isolated
from both coastal and oceanic waters in recent years (Yurkov
& Beatty, 1998). Furthermore, new pigmented species, such
as Bacillus firmus, which used to be regarded as colourless,
have been found in marine environments (Pane et al., 1996;
Siefert et al., 2000). Carotenoids harvest light of wavelength
between 460 and 550 nm, which can penetrate to c. 200 m
depth in clear oligotrophic oceanic waters, a greater depth
than in eutrophic coastal waters (50 m or more) (Ackleson,
2003). Moreover, the absorbency values of some pigmented
FEMS Microbiol Ecol 57 (2006) 92–105
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Diversity and distribution of pigmented bacteria
heterotrophic bacteria (PHB) measured under various controlled conditions of light and nutrients showed that the
values of PHB in the blue spectral region could be at least
twice, or up to one order of magnitude higher than, those of
non-pigmented heterotrophic bacteria (NHB), suggesting
the high potential of PHB in terms of the utilization of light
energy (Stramski & Kiefer, 1998).
The functions of carotenoids in photosynthetic organisms are to provide photooxidative protection and to
transfer the light quanta to the photosynthetic reaction
centre. For nonphotosynthetic bacteria, carotenoids offer
similar photooxidative protection against other photosensitizing porphyrin molecules, such as protoporphyrin IX and
heme (Armstrong, 1997). Carotenoid-containing Grampositive bacteria, such as Staphylococcus lutrae and Staphylococcus aureus, are more resistant to the lethal effects of gas
phase 1O2 than their colourless mutant strains (Dahl et al.,
1989). A field investigation has shown that the pigmented
heterotrophic bacteria exhibit a higher degree of multiple
drug resistance than nonpigmented strains (Egan et al.,
2002a,b; Hermansson et al., 1987), and a positive correlation
between the antifouling activities and the expression of
pigment was found in Pseudoalteromonas tunicata strain
D2. No antifouling activity, and less expression of pigment,
is observed when the strain grows on nutrient-rich medium,
indicating that the expression of carotenoid is inhibited by
organic-rich nutrient (Egan et al., 2002a). All these findings
provide a new clue to probing the ecological functions of
bacterial carotenoids in marine environments. In view of the
strong light absorption of carotenoids in the near-blue
spectral region, the heterotrophic bacteria may contribute
more to marine light absorption and light energy transformation than previously thought.
Despite the great progress that has been made in understanding the functions of bacterial carotenoids, the natural
distribution and genetic diversity of PHB in marine environments remain unclear. In this study, we applied both the
cultivation method and 16S rRNA gene sequence analysis to
investigating the horizontal and vertical distributions and
genetic diversity of PHB in coastal, shelf and oceanic waters.
We have gained new insights into the environmental properties that control distribution, and reveal the high diversity of
PHB for the first time.
Materials and methods
Sampling sites
We sampled surface and subsurface (10–30 m depth) water
in the eutrophic Yangtze River Estuary, mesotrophic East
China Sea, South China Sea and oligotrophic North Pacific
Ocean, including four vertical profiles. Sampling locations
and dates are shown in Table 1. Sea-Bird 911 plus was used
FEMS Microbiol Ecol 57 (2006) 92–105
for CTD (depth, temperature, salinity, density), with auxiliary inputs (epi-fluorescence and dissolved oxygen) for
water column recording. Seawater was sampled with Niskin
bottles at various water depths, according to the CTD
profiles.
Enumeration of total bacteria
Aliquots of 2 mL of seawater were fixed for 15 min with 1%
paraformaldehyde (PFA) immediately after sampling and
then stored at 20 1C for later analysis. Total heterotrophic
bacterial abundance (TB) was determined by flow cytometry
(Marie et al., 1997).
Enumeration of CFU and PHB
Subsamples of 100 mL of fresh seawater were spread onto
Marine 2216E agar medium (Zobell & Morita, 1957)
modified using 0.1 g L1 peptone and 0.05 g L1 yeast extraction. The plates were initially incubated under the in situ
temperature of the corresponding sampling station in the
dark for 7 days to eliminate oxygenic photoautotrophs, and
then placed under a natural light/dark cycle (12 h/12 h) for
the formation of pigmented colonies (Koblizek et al., 2003).
Peptone, yeast extraction and microelements in 2216E
medium were purchased from Amersco (Solon, OH), and
macroelements from Sangon Co. Ltd (Shanghai, China).
The total and pigmented colonies were counted after two
weeks of incubation. The isolates were streaked out and
transferred several times to obtain pure cultures. The pure
cultures were stored in tubes at 4 1C for immediate use, and
at 20 1C with 30% glycerol added for long-term storage.
All types of pigmented colonies were picked randomly.
16S rRNA gene sequencing and phylogenetic
analysis
Genomic DNA of isolates were prepared according to
Wisotzkey et al. (1990) with minor modifications; that is,
1% sodium dedecyl sulphate (SDS) was used to denature
protein instead of Proteinase K digestion. 16S rRNA genes
were amplified using a eubacterial primer, SSEub27F
(5 0 -AGAGTTTGATCATGGCTCAG-3 0 ) (Giovannoni et al.,
1988), and a universal primer, SS1492R (5 0 -GGTACCTTGTTACGACTT-3 0 ) (Lane, 1991), corresponding to the positions 8–27 and 1492–1501 respectively on the Escherichia
coli 16S rRNA gene. The nearly full-length sequence of the
16S rRNA gene (c. 1465 bp) was amplified on a T3 thermocycler (Biometra Co., Göttingen, Germany). The reaction
conditions were as follows: initial denaturation at 94 1C for
4 min, followed by 30 cycles of 94 1C for 1 min, 55 1C for
1 min, and 72 1C for 2 min, with a final extension step at
72 1C for 8 min. The PCR products were gel-purified using a
Gel Extraction Kit (TaKaRa Biotechnology Co. Ltd, Dalian,
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H. Du et al.
Table 1. Sampling stations, site locations, depths and dates
Site location
Station
Yangtze River estuary
DA4
DC10
DE5
DF22
DF23
DG28
South China Sea
D1
D6
B3
B5
East China Sea
p3
p4
p6
p8 (70 m)
P9
A6
A5
A4
A3
A2
A1
S1
P10
North Pacific Ocean
pf1 (1500 m)
pf2 (2000 m)
pf3 (3000 m)
dy-h17
dy-h15
dy-h14
Latitude, longitude
Sampling depth (m)
Sample date
123.5E, 32.0N
122.5E, 31.0N
123.0E, 29.5N
122.5E, 29.5N
123.0E, 29.5N
123.5E, 29.0N
0, 20
0, 20
0, 20
0, 20
0, 20
0, 20
09/08/2003
10/08/2003
11/08/2003
15/08/2003
16/08/2003
21/08/2003
113.5E, 18.5N
111.0 E, 21.1N
116.0E, 21.5N
115.1E, 22.2N
0, 25
0, 25
0
0
19/02/2004
17/02/2004
01/03/2004
01/03/2004
127.4E, 28.4N
126.8E, 28.6N
126.2E, 29.0N
125.0E, 29.6N
124.0E, 30.1N
128.9E, 31.5N
127.8E, 29.5N
128.0E, 29.9N
128.4E, 30.5N
128.6E, 30.9N
128.8E, 31.5N
129.1E, 32.0N
123.5E, 30.5N
0, 20
0, 20
0, 20
0, 20, 45, 50, 65
0, 15
0, 25
0, 25
0, 20
0, 30
0, 30
0, 30
0, 30
0, 15
14/09/2003
14/09/2003
13/09/2003
13/09/2003
13/09/2003
14/09/2003
14/09/2003
15/09/2003
15/09/2003
15/09/2003
15/09/2003
23/09/2003
13/09/2003
103.3W, 12.5N
104.0W, 12.4N
117.3W, 13.2N
109.3W, 13.0N
113.5W, 13.2N
120.7W, 13.3N
0.5,10,30,50,75,100,125,150,175,200
0.5,10,30,50,75,100,125,150,175,200
0.5,10,30,50,75,100,125,150,175,200, 500, 1000, 1500, 2850
0
0
0
01/11/2003
03/11/2003
02/11/2003
03/11/2003
02/11/2003
31/10/2003
Station for vertical profile investigation (total depth indicated in parentheses).
China; Code No. DV805A), according to the manufacturer’s
instructions. Ligation into pMD18-T vector and transformation into E. coli DH5a were performed according to the
manual of products (TaKaRa; Code No. D504A). Colony
PCR was done by toothpicking an ampicillin-resistant single
colony in a 25 mL PCR tube for screening target inserts with
pMD18-T vector sequencing primers M13-47 (5 0 CGCCAGGGTTTTCCCAGTCACGAC-3 0 ) (TaKaRa, Code
No. D3887) and RV-M (5 0 -AGCGGATAACAATTTCACACAGG-3 0 ) (TaKaRa, Code No. D3880). The 16S rRNA gene
heterogeneity was tested using restriction fragment length
polymorphism (RFLP) based on restriction enzymes AfaI
and HhaI (TaKaRa). The recombinant plasmid DNA was
extracted and sequenced on ABI 377A automated sequencer
(Applied Biosystems, Foster City, CA) using the sequencing
primers M13-47 and RV-M of the vector, and a third primer
was designed based on the sequenced fragment for achieving
the complete sequence. The near-full-length nucleotide
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sequences of 16S rRNA gene (c. 1465 bp) were analysed
using DNASTAR Editseq software (version 5.0 Inc., Madison, WI). Sequences from the current study, combined with
the most homologous reference sequences retrieved from
the NCBI database (http://www.ncbi.nlm.nih.gov/), were
aligned using the Clustal W method in the package (DNASTAR). The genetic heterogeneity of the sequences was
evaluated by percent identities using DNASTAR software
(version 5.0 Inc.). A neighbour-joining analysis (Saitou &
Nei, 1987) was used to reconstruct phylogenetic trees using
the MEGA program (Kumar et al., 2004). A bootstrap
analysis (100 replicates), using outgroup species that were
well chosen according to their phylogenetic relatives, was
performed to evaluate the topology of the phylogenetic tree.
The 16S rRNA gene sequences obtained from this study were
deposited in GenBank with accession numbers
AY745813–AY745871; AY646155–AY646165; DQ073100–
DQ073103. All isolates belonging to Alphaproteobacteria
FEMS Microbiol Ecol 57 (2006) 92–105
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Diversity and distribution of pigmented bacteria
Vertical profiles of TB, CFU and PHB
were screened for the presence of bacteriochlorophyll by
high-performance liquid chromatography and for the presence of the pufM gene that encodes the M subunit of the
reaction center complex of photosynthesis by PCR, according to a previous study (Koblizek et al., 2003).
The average data of vertical profiles (from surface to 200 m
depth) of TB, CFU and PHB at three stations (pf1, pf2 and
pf3) in the tropical North Pacific Ocean are shown in Fig. 1.
The abundance of TB and CFU decreased with water depth,
with the highest abundance (8.3 1.1 105 cell mL1 for
TB, 6.2 0.9 103 cell mL1 for CFU) occurring at 10 m
depth (Fig. 1a, b). The abundance of PHB (1751 102
cell mL1) and the ratio of PHB to CFU (31.2 2.5%) were
highest in surface water, showing no significant difference
from the surface to 30 m depth, and then decreased sharply
with increasing water depth (Fig. 1e, f).
The complete vertical profiles at station pf3 and station
P8, two distinct water columns, were investigated (Fig. 2). At
station pf3 (3000 m water column), TB and CFU were
present throughout the whole water column, whereas PHB
only occupied the euphotic zone (200 m) and were predominant in the high-light layer, with an average of 1.6 103
cell mL1 in the water column above 30 m. PHB still existed
in the water column from 50 to 125 m, with an average
abundance of 250 cell mL1, but completely disappeared
below 500 m (Fig. 2a). Correspondingly, the concentrations
of chlorophyll a were detectable in the water column down
to 200 m, with the maximum value occurring at 30 m (Fig.
2c). The highest abundance of TB (6.6 105 cell mL1) and
CFU (5.8 103 cell mL1) occurred in the 30 m layer, and
decreased with increasing depth (Fig. 2a). In terms of the
CFU/TB profile, peaks emerged at 30 m (0.92%), 125 m
(0.52%) and 50 m from the bottom (0.53%), with the
minimum value (0.13%) at 500 m depth (Fig. 2b), while
the PHB/CFU ratio showed a sharp decrease below 50 m
(Fig. 2c). At station P8 (70 m water column), TB, CFU and
PHB were found in the whole water column (Fig. 2d), and
their abundance and the value of CFU/TB were higher than
in Pacific Ocean water. The ratio of CFU to TB peaked at
20 m (4.22%) and 65 m (3.21%) depth (near bottom) (Fig.
2e), with the minimum value appearing at 45 m (2.01%).
However, the ratio of PHB to CFU decreased sharply from
Analysis of pigment in acetone--methanol
extracts
Isolates were incubated in liquid 2216E medium at 20 1C
with a light cycle of 12 h/12 h. Cells were harvested by
centrifugation at 1073 g for 5 min. Pigments were extracted
from the cells with acetone–methanol (7 : 2, volume in
volume) at 4 1C for 12 h in dark. Absorbance data (OD)
from 200 to 900 nm were collected using a UV–Vis–
NIR spectrophotometer (Cary 50, Varian Co, Palo Alto,
CA) with a slit width of 2 nm.
Results
Distribution of PHB
Spatial distribution of CFU and PHB
The abundances of TB and CFU decreased from eutrophic
coastal regions to the oligotrophic eastern tropical North
Pacific Ocean, and increased from surface to subsurface
water (Table 2). The maximum values of TB and CFU were
observed in the subsurface layers (c. 10–30 m depth) of the
Yangtze River Estuary, where CFU made up a significant
fraction of the total bacterial count with maximum culturability (6.12%). In this sampling site, the greatest abundance
of PHB (9.9 103 cell mL1) and ratio of PHB to CFU
(39.60%) were also found in surface water, and then
decreased in the subsurface. However, no distinct difference
between surface (31.42%) and subsurface (28.57%) ratios
was found in the eastern tropical North Pacific Ocean
(Table 2).
Table 2. Abundance of total heterotrophic bacteria (TB), CFU, and pigmented heterotrophic bacteria (PHB) in various marine environments
Yangtze River Estuary
Subsurfacez
Surface‰
1
TB (cells mL )
CFU (cells mL1)
PHB (cells mL1)
PHB/CFU (%)
CFU /TB (%)
East China Seaw
5
7.1 10
2.5 104
9.9 103
39.60
3.52
5
8.2 10
5.1 104
9.8 103
19.24
6.12
Eastern Tropical North Pacific Oceanz
Subsurfacez
Surface‰
5
5.9 10
1.2 104
4.2 103
35.00
3.43
Subsurfacez
Surface‰
5
5
6.1 10
1.8 104
3.1 103
17.22
4.28
4.8 10
3.5 103
1.1 103
31.42
0.72
5.7 105
4.2 103
1.2 103
28.57
0.73
Averaged over six stations in the Yangtze River Estuary.
w
Averaged over 13 stations in the East China sea.
Averaged over three stations in the Eastern Tropical North Pacific Ocean.
‰
Samples from 0 to 1 m depth.
z
Samples from 10 to 30 m depth.
z
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96
H. Du et al.
0
2e+5
cell mL−1
4e+5
6e+5
8e+5
1e+6 0
(a)
cell mL−1
1e+3 2e+3 3e+3 4e+3 5e+3 6e+3 7e+3
0
(b)
1e+3
cell mL−1
2e+3
3e+3
(c)
Water depth (m)
0
50
100
150
200
CFU
TB
PHB
250
%
1
0
2
(d)
0.0
0.1
%
0.2
0.3
(e)
0.4
0
5
10
15
%
20
25
30
35
40
(f)
Water depth (m)
0
50
100
150
200
CFU/TB%
PHB/CFU%
PHB/TB%
250
Fig. 1. Depth profiles of (a) total bacteria (TB), (b) CFU, (c) pigmented heterotrophic bacteria PHB, and the ratios of (d) CFU to TB, (e) PHB to TB and (f)
PHB to CFU in the eastern Tropical North Pacific. Data were averaged over the 3 stations pf1, pf2 and pf3.
surface to bottom, showing a similar profile to the light
intensity attenuation (Fig. 2f).
16S rRNA gene sequences analysis
The 16S rRNA genes of 247 pigmented isolates were
analysed using PCR-RFLP. According to the RFLP profiles,
nearly full-length sequences of 74 isolates were sequenced (c.
1465 bp). Isolates with sequences of more than 98% similarity were grouped into the same operational taxonomic unit
(OTU) based on sequences analysis. All the isolates showed
various colours with genus or species specificity, including
pink, red, golden, orange, yellow and buff. For example, all
isolates belonging to the genus Erythrobacter showed buff,
while isolates JL-64 and JL-65 belonging to Paracoccus
exhibited yellow and red, respectively. The majority of the
isolates belonging to Exiguobacterium displayed bright yellow, e.g. Yangtze River Estuary (YGE)-JL-47–49, and ECSJL-36, with the rest in bright red or orange. The RFLP
analysis revealed that several colourless isolates were af-
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filiated to the genera Pseudoalteromonas, Halomonas, Marinomonas and Bacillus.
The 16S rRNA gene analysis provided evidence for spatial
variability of the bacterial community structure of PHB.
Most PHB belonging to Alphaproteobacteria and Bacteroidetes were obtained mainly from the South China Sea and
East China Sea. PHB isolates from the eastern tropical North
Pacific Ocean were predominantly affiliated to Gammaproteobacteria (78.7%), while most isolates from the Yangtze
River Estuary clustered into Actinobacteria (20.5%) and
Firmicutes (71.8%) (Table 3). The phylogenetic trees show a
wide phylogenetic heterogeneity of PHB, with all the 35
OTUs affiliated to 25 genera of six phylogenetic classes,
including Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria (Fig. 3a–e).
Alphaproteobacteria group (Fig. 3a)
Sixty one out of 247 isolates were affiliated to two
families, Rhodobacteraceae and Sphingomonadaceae, of
Alphaproteobacteria, including four OTUs that belong to
FEMS Microbiol Ecol 57 (2006) 92–105
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Diversity and distribution of pigmented bacteria
cell mL−1
%
PHB, CFB
0 1e+3 2e+3 3e+3 4e+3 5e+3 6e+3 7e+3
0.0
(a)
0
0.2
0.4
%
0.6
0.8
1.0
0
(b)
5
10
15
20
25
30
35
(c)
50
100
Water depth (m)
150
200
250
500
1000
1500
TB
CFU
PHB
2000
2500
PHB/CFU %
Chl α ug / L
CFU/ TB %
3000
0 1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 7e+5
TB
0
1e+4
PHB, CFB
2e+4
3e+4
4e+4
0.00
1
(d)
2
3
4
5
0
(e)
0.05
5
0.10
0.15
ug / L
10
15
20
0.20
0.25
25
30
(f)
Water depth (m)
0
20
40
CFU/TB %
TB
CFU
PHB
60
PHB/ CFU %
I0%
1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 7e+5 8e+5
0
20
40
60
80
100
120
I0%
TB
Fig. 2. Whole water-column depth profiles of total bacteria (TB), CFU and pigmented heterotrophic bacteria (PHB) abundance (a, d), ratio of CFU to TB
(b, e), and ratio of PHB to CFU (c, f) at station pf3 in the Pacific Ocean (a, b, c), and at station P8 in the East China Sea (d, e, f). I1%, percentage of surface
light intensity.
the genera Ruegeria, Paracoccus and Erythrobacter and a
distinct cluster ECS-JL-137 (ECS-JL-137, -135, -132, -131, 129). The closest relative of this cluster is an unidentified
Rhodobacteraceae bacterium with a lower similarity of
94.4%. Thus, these isolates are not clearly affiliated to any
genus based on 16S rRNA gene analysis and may be a novel
cluster or species that needs to be identified further. PHB
related to Ruegeria were obtained from the East China Sea,
while those related to Erythrobacter were obtained from the
South China Sea (Fig. 3a). No Bacteriochlorophyll or pufM
Table 3. Frequency and phylogenetic affiliation of pigmented heterotrophic bacteria based on PCR-restriction fragment length polymorphism
combined with 16S rRNA gene sequence analysis
Yangtze River Estuary
South China Sea
East China Sea
Eastern Tropical North Pacific
Group
Total isolates
Isolates
%
Isolates
%
Isolates
%
Isolates
%
Alphaproteobacteria
Gammaproteobacteria
Bacteroidetes
Actinobacteria
Firmicutes
S
61
76
11
37
62
247
5
1
0
16
56
78
8.2
1.3
0
43.2
90.3
31.6
20
8
9
10
1
48
32.8
10.5
81.8
27.0
1.6
19.4
25
8
1
8
4
46
40.9
10.5
9.1
21.6
6.4
18.6
11
59
1
3
1
75
18.0
77.6
9.1
8.1
1.6
30.3
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H. Du et al.
genes were detected in any of the isolates belonging to the
above clusters. Three isolates, NPO-JL-65, SCS-JL-S11 and
NPO-JL-64, showed close similarity (above 98.5%) with
Paracoccus marcusii (Baj, 2000) and exhibited bright colours
of red, pink and yellow, respectively.
Gammaproteobacteria group (Fig. 3b)
Compared with the Alphaproteobacteria group, the Gammaproteobacteria group showed a higher genetic diversity. In
total, 76 isolates were isolated, mainly from the North
Pacific Ocean (59 isolates), South China Sea (8 isolates)
and East China Sea (8 isolates). The phylogenetic tree
encompassed two principal clades, one containing eight
OTUs affiliated to the four genera Pseudoalteromonas,
Vibrio, Alteromonas and Shewanella, and the other contain-
ing 4 OTUs belonging to the genera Deleya, Halomonas,
Marinomonas and Pseudomonas. The isolates of OTUs
Pseudoalteromonas (35 isolates) and Halomonas (22 isolates)
together occupied 75% of isolates of this group that were
mainly obtained from the North Pacific Ocean, South China
Sea and East China Sea. Ten Alteromonas-related isolates
were all from the South China Sea, and three Vibrio
calviensis-related isolates were from the East China Sea. A
few isolates belonging to the genera Deleya, Pseudomonas
and Shewanella were obtained from the North Pacific Ocean.
Actinobacteria group (Fig. 3c)
The Actinobacteria consisted of three families, Micrococcaceae, Nocardiaceae and Dietziaceae, including four genera,
namely Kocuria, Micrococcus, Rhodococcus and Dietzia. Only
100
(a)
0.02
95
97
99
78
ECS-JL-137 (AY646164)
ECS-JL-135 (AY745857)
ECS-JL-132 (AY745856)
ECS-JL-131 (AY646163)
ECS-JL-129 (AY646161)
Rhodobacteraceae bacterium (AY442178)
Hydrothermal vent strain TB66 (AF254109)
Salipiger mucescens (AY527274)
Ruegeria algicola (X78314)
84
Ruegeria sp.DG898 (AY258086)
98
ECS-JL-126 (AY745859)
69
Roseobacter sp.TM1040 (AY332662)
Ruegeria atlantica (AF124521)
Roseobacter litoralis (AJ012707)
59
100 Paracoccus marcusii (Y12703)
Paracoccus carotinifaciens (AB006899)
NPO-JL-65 (AY745834)
90
SCS-JL-S11 (AY745863)
89
NPO-JL-64 (AY646160)
Paracoccus denitrificans (AY157621)
91 SCS-JL-310 (AY646156)
SCS-JL-316 (AY646157)
65
Erythrobacter citreus (AF227259)
Erythrobacter vulgaris (AY706938)
82 SCS-JL-S3 (AY745821)
SCS-JL-S4 (AY745820)
63
Erythrobacter flavus (AF500005)
88
Erythrobacter gaetbuli (AY562220)
100 Porphyrobacter neustonensis (AB033327)
78
Porphyrobacter tepidarius (AB033328)
Erythrobacter litoralis (AB013354)
Pseudomonas putida (AB109776)
92
78
Fig. 3. Phylogenetic relationships of 16S rRNA gene sequences of pigmented heterotrophic bacteria. (a) Alphaproteobacteria group, outgroup:
Gammaproteobacteria species Pseudomonas putida; (b) Gammaproteobacteria group, outgroup: Alphaproteobacteria species Roseobacter litoralis;
(c) Actinobacteria group, outgroup: Firmicutes bacteria Bacillus catenulatus; (d) Firmicutes group, outgroup: Actinobacteria bacterium Rhodococcus
fascians; and (e) Bacteroidetes, outgroup: Alphaproteobacteria Roseobacter litoralis. The trees were constructed with the Clustal W program using
the neighbor-joining algorithm. A mask of 1460 nucleotide positions was used to construct the tree. Bootstrap analysis of one hundred replicates
was performed. Values of 450% are shown on the nodes. The bar corresponds to base substitutions per 100 nucleotide positions. SCS-JL-XXX, ECSJL-XXX, YGE-JL-XXX and NPO-JL-XXX represent isolates from the South China Sea, East China Sea, Yangtze River Estuary and North Pacific
Ocean, respectively.
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FEMS Microbiol Ecol 57 (2006) 92–105
99
Diversity and distribution of pigmented bacteria
(b)
NPO-JL-58 (AY745828)
NPO-JL-54 (AY745825)
Pseudoalteromonas haloplanktis (AF214729)
88 Gamma proteobacterium UMB20C (AF505745)
84 Pseudoalteromonas sp . D20 (AY582936)
98
NPO-JL-62 (AY745832)
Pseudoalteromonas chazhmella (AY682201)
96
NPO-JL-300 (AY646155)
100 ECS-JL-96 (AY745871)
78
Pseudoalteromonas sp . AS-43 (AJ391204)
56 SCS-JL-S1(AY745839)
87
Pseudoalteromonas spongiae (AY769918)
98
Vibrio sp.SG128 (AB038027)
76
Vibrio calviensis (AF118021)
ECS-JL-73 (AY745814)
78
Vibrio hollisae (AJ514911)
Photobacterium phosphoreum (AY435156)
Alteromonas alvinellae (AF288360)
95
92 Alteromonas macleodii (AMY18228)
66
96
SCS-JL-S9 (AY745861)
100 SCS-JL-S12 (AY745818)
SCS-JL-S5 (AY745819)
99 NPO-JL-56 (AY745827)
Shewanella livingstonensis (AY771775)
Shewanella frigidimarina (U85902)
NPO-JL-63 (AY745827)
SCS-JL-S8 (AY745837)
100
Deleya pacifica (L42616)
NPO-JL-59 (AY745829)
80
ECS-JL-104 (AY745860)
89
ECS-JL-81 (AY745870)
82
Halomonas marina (AJ306890)
Halomonas halocynthiae (AJ417388)
68
99 NPO-JL-55 (AY745826)
Marinomonas sp.BSW10005 (AY646429)
76
Marinomonas pontica (AY539835)
89
NPO-JL-67 (AY745835)
Pseudomonas putida(AB109776)
Roseobacter litoralis (AJ012707)
100
0.02
59
(c)
0.02
68
66
100 ECS-JL-75 (AY745841)
YGE-JL-51 (AY745851)
ECS-JL-70 (AY745840)
88 100 YGE-JL-40 (AY745845)
ECS-JL72 (AY745813)
78
Kocuria palustris (Y16263)
Kocuria rhizophila (Y16264)
Kocuria polaris (AJ278868)
99 YGE-JL-76 (AY745846)
95
Micrococcus sp.TUT1210 (AB188213)
Micrococcus lylae (AF057290)
Rhodococcus fascians (AJ011329)
100
98 NPO-JL-60 (AY745830)
92
YGE-JL-61 (AY745831)
Rhodococcus yunnanensis (AY602219)
89
Rhodococcus wratislaviensis (AY940038)
100 SCS-JL-S2 (AY745838)
Rhodococcus equi (X80614)
88 SCS-JL-S7 (AY745816)
Dietzia maris (AB211032)
Dietzia natronolimnaea (AJ717373)
Bacillus catenulatus (AY523411)
78
Fig. 3. Continued.
FEMS Microbiol Ecol 57 (2006) 92–105
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c
100
H. Du et al.
100 YGE-JL-26 (AY745824)
YGE-JL-31 (AY745869)
79
Bacillus kangii (AF281158)
YGE-JL-29 (AY745867)
95
71
Bacillus aquaemaris (AF483625)
88
NPO-JL-69 (AY745836)
Planococcus kazaiensis (AY260168)
98
Planococcus psychrophilus (AJ314746)
100
82
Planococcus mcmeekinii (AF041791)
Bacillus arsenicus (AJ606700)
99
86
Bacillus barbaricus (AJ422145)
ECS-JL-74 (AY745842)
Bacillus gelatini (AJ586347)
57
100 Bacillus indicus (AJ583158)
100 Bacillus catenulatus (AY523411)
95 YGE-JL-44 (AY745847)
YGE-JL-39 (AY745844)
92
YGE-JL-38 (AY745843)
68
YGE-JL-78 (AY745849)
82
YGE-JL-34 (AY745866)
Bacillus horikoshii (AB043865)
99 YGE-JL-35 (AY745864)
95 YGE-JL-42 (AY745848)
YGE-JL-25 (AY745823)
82
Exiguobacterium sp.BTAH1 (AY205564)
ECS-JL-36
(AY745858)
94
YGE-JL-52 (AY745855)
Exiguobacterium aestuarii (AY594265)
YGE-JL-31(AY745869)
89
YGE-JL-47 (AY745850)
100 YGE-JL-48 (AY745851)
68
92 YGE-JL-49 (AY745852)
Exiguobacterium lactigenes (AY818050)
76 YGE-JL-24 (AY745822)
Exiguobacterium marinum (AY594266)
Rhodococcus fascians (AJ011329)
(d)
98
0.02
64
(e)
0.02
100 SCS-TW-17 (DQ073101)
SCS-TW-80 (DQ073100)
Gramella portivictoriae (DQ002871)
Gramella
echinicola
(AY608409)
89
86
Flavobacterium salegens (M92279)
66 92
Salegentibacter flavus (AY682200)
SCS-TW-20(DQ073102)
Cytophaga marinoflava (AY167315)
84
SCS-JL-S6 (AY745817)
Psychroserpens burtonensis (AY771714)
76
68
SCS-TW-49 (DQ073103)
Winogradskyella poriferorum (AY848823)
Bacteroidetes bacterium (AF539755)
96 Flavobacteriaceae bacterium (AY353813)
92
Polaribacter irgensii (AY771712)
64
Polaribacter franzmannii (U14586)
87 Polaribacter dokdonensis (DQ004686)
SCS-JL-S10 (AY745862)
Roseobacter litoralis (AJ012707)
98
58
90
Fig. 3. Continued.
three out of 37 isolates were obtained from the eastern
tropical North Pacific Ocean. All isolates related to the genus
Kocuria exhibited lemon yellow, while isolates belonging to
Rhodococcus exhibited salmon pink.
Firmicutes group (Fig. 3d)
This group comprised the predominant PHB that were
isolated from the Yangtze River Estuary. The phylogenetic tree
encompassed two clades, one containing Bacillus and Planococcus genera, and the other containing representative species
of the genus Exiguobacterium, including the seven OTUs
Bacillus kangii, Bacillus aquaemaris, Planococcus, Bacillus
arsenicus, Bacillus catenulateus, Bacillus horikoshii, and Ex2006 Federation of European Microbiological Societies
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c
iguobacterium (Fig. 3d). The Bacillus clade comprised a
phylogenetically and phenotypically heterogeneous group of
species, including six OTUs with a distant affiliation with each
other (similarity below 95.5%). These isolates showed speciesspecific colour; for example, isolates YGE-JL-38, -29 and -74
were orange, while YGE-JL-39, -44 and -78 were yellow. All
isolates belonging to the genus Exiguobacterium were obtained from the Yangtze River Estuary and displayed different
colours, varying from primrose yellow to saffron yellow.
Bacteroidetes group (Fig. 3e)
In this group, six sequenced isolates were clustered into the
two families of Flavobacteriaceae and Flexibacteraceae, with a
FEMS Microbiol Ecol 57 (2006) 92–105
101
Diversity and distribution of pigmented bacteria
Analysis of absorption spectra of pigment
Pigments in acetone extracts of some representative isolates
were detected using a spectrophotometer, and the data of
four examples are shown in Fig. 4. The light of wavelength
between 450 and 550 nm was most strongly absorbed,
corresponding to their inherent colour. The absorption
spectra also revealed species or genus specificity. For example, acetone extracts of representative isolates NPO-JL-64
and NPO-JL-65 of the Alphaproteobacteria group exhibited
one peak at 495 and 462 nm, respectively. Pigment extracts
of isolates NPO-JL-59 (Gammaproteobacteria) and YGE-JL42 (Firmicutes) displayed similar absorption spectra, with
three peaks at 470, 499 and 527 nm for NPO-JL-59, and 475,
501 and 531 nm for YGE-JL-42.
Discussion
This study has revealed, for the first time, the distribution
patterns and genetic diversity of PHB in a variety of marine
environments based on a culture-dependent approach and
16S rRNA gene sequence analysis. The culturability of hererotrophic bacteria on Marine agar 2216E medium containing a low amount of organic carbon varied from 0.7% for
the oligotrophic Pacific Ocean to 6.1% for the eutrophic
estuary. Previous reports have also revealed that the culturability of bacteria varies with peptone concentration in the
medium and source of samples, whereas pigmented colonies
are easier to grow on low-peptone media (Buck, 1974).
Investigation of lakes showed that more CFU can be
obtained in nutrient-rich waters than in nutrient-poor
waters (Porter et al., 2004).
Abundance distribution of PHB in marine
environments
Although the horizontal distributions of the abundance of
CFU and PHB showed similar patterns, the PHB predominated in the euphotic zone in term of vertical distribution,
and showed a close association with light (Figs 1 and 2). The
ratio of PHB to CFU in the surface layer was nearly twice
that in subsurface layers in turbid estuaries, while it differed
FEMS Microbiol Ecol 57 (2006) 92–105
0.08
NPO-JL-59
0.07
OD
high genetic heterogeneity because of the low percent
identities (c. 82.6–89.5%) of the 16S rRNA gene sequences
with each other. Four isolates (SCS-TW-17, -80, -20 and
-49) were not clearly affiliated to a known genus based on
16S rRNA gene analysis. Furthermore, their 16S rRNA gene
similarities with the most homologous species were below
95.8%, indicating that novel species of Bacteroidetes inhabit
our sampling sites (Fig. 3e). These isolates displayed various
colours with species-specificity; e.g. SCS-TW-20 showed
pink, SCS-TW-49 showed yellow, and SCS-TW-17 and
SCS-TW-80 showed buff.
YGE-JL-42
0.06
NPO-JL-65
0.05
NPO-JL-64
0.04
0.03
0.02
0.01
0
400
450
500
550
Wavelength (nm)
600
650
Fig. 4. Absorption spectra of pigments in acetone extracts of isolate
cells.
little between surface and subsurface water in the clear
eastern tropical North Pacific Ocean. The complete vertical
profile of the PHB/CFU ratio displayed a similarity with the
light intensity attenuation, and few pigmented isolates were
found in deep seawater (from 500 m to bottom) in this
study. These phenomena suggest that, in the upper layer of
the euphotic zone, with strong light radiation, PHB have the
advantage of a carotenoid-related light-protection mechanism, because carotenoids can reduce the damage of photooxidative and UV radiation (Hermansson et al., 1987;
Sandmann et al., 1998; Egan et al., 2002a). It has been
reported that bacteria living in extreme conditions (very low
or high temperatures, high salinity, acidic conditions, strong
light, etc.) have adopted carotenoids suitable for membrane
stabilization of the cell wall (Yokoyama et al., 1996). Here,
PHB account for up to 39.6% of culturable bacteria, and
1.4% of the total microbial community was revealed, which
might even be an underestimate because it is probable that
not all pigmented bacteria were isolated.
Genetic diversity of PHB
A wide genetic diversity of marine PHB varying with
environmental factors has been revealed. Genetic analysis
suggests that the distribution of PHB components is related
to water masses in the ocean and is controlled by their
environmental and biogeochemical properties. Pigmented
representatives were found in all of the following groups:
Alphaproteobacteria, Gammaproteobacteria, Actinobacteria,
Firmicutes and Bacteroidetes.
The sequences affiliated with the Alpha and Gamma
subclasses of Proteobacteria are commonly retrieved from
marine aquatic ecosystems, and the Alpha subclass is more
abundant in seawater than in freshwater (Glockner et al.,
1999). Roseobacter and Erythrobacter, which belong to the
Alpha-3 Proteobacteria and Alpha-4 Proteobacteria subclasses, are characterized by their abundant production of
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102
carotenoids (Yurkov & Beatty, 1998). Roseobacter is one of
the largest clades affiliated to the Alpha subclass of Proteobacteria that comprise a large fraction of heterotrophic
marine bacteria and shows environmental properties controlling distribution pattern (Selje et al., 2004). Pigmented
Erythrobacter species are also often obtained from various
marine environments, including coastal, shelf and open
ocean waters (Yurkov & Beatty, 1998; Koblizek et al., 2003).
Most Roseobacter and Erythrobacter strains display bright
colours, and include aerobic anoxygenic photosynthesis
phenotypes and nonphotosynthesis phenotypes (Yurkov &
Beatty, 1998; Denner et al., 2002; Allgaier et al., 2003). The
functions of carotenoids, which comprise a diverse class of
pigments found in photosynthetic and nonphotosynthetic
organisms, are protection from photooxidative damage and
in light absorption, and as a structural component of the
photosynthetic membranes in anoxygenic phototrophic
bacteria (Cogdell & Frank, 1987). In this study, no BChl a
or pufM genes were detected from those pigmented isolates
related to Roseobacter-like cluster (ECS-JL-126) and the
proposed novel cluster (ECS-JL-137, -135, -132, -131, -129)
belonging to the family Rhodobacteraceae, or to Erythrobacter clade (SCS-JL-310, -316, -S3 and -S4). Thus the
difference in carotenoid function in the two photosynthetic
and nonphotosynthetic phenotypes needs to be studied
further.
It has been reported that traditional cultivation methods
have attributed a high importance to some members of the
Gammaproteobacteria, while fluorescence in situ hybridization data have revealed that it is a minor component (o4%)
of the bacterioplankton (Eilers et al., 2000). Moreover,
representative species of the genera Pseudoalteromonas,
Alteromonas and Vibrio are considered as readily culturable
bacteria (Eilers et al., 2000). In this study, 64 out of 76
Gammaproteobacteria pigmented isolates belonged to the
above three genera, and 42 Pseudoalteromonas isolates
showing bright colours were mostly from the eastern
tropical North Pacific Ocean. Numerous marine species of
the genus Pseudoalteromonas have attracted significant interest because of their association with marine eukaryotic
hosts (Lovejoy et al., 1998; Holmstrom & Kjelleberg, 1999)
and the production of bioactive compounds that exhibit
antibacterial, algicidal, antifungal, agarolytic, and antiviral
activities (Lovejoy et al., 1998; Holmstrom & Kjelleberg,
1999; Egan et al., 2002a, b; Holmstrom et al., 2002;
Isnansetyo & Kamei, 2003). Moreover, it has been revealed
that the antifouling capability of Pseudoalteromonas tunicata
is positively correlated with its yellow pigmentation (Egan
et al., 2002a). In the Gammaproteobacteria group, representatives of the genera Pseudomonas, Halomonas and Marinomonas have seldom been screened by culture-dependent
methods, and their abundances in marine environments
have been unclear.
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H. Du et al.
The Actinobacteria bacteria are primarily saprophytic,
and are best known from soils, where they contribute
significantly to the turnover of complex biopolymers such
as lignocellulose, hemicellulose, pectin, keratin, and chitin
(Williams et al., 1984; Stackebrandt et al., 1997). Thus it has
frequently been assumed that actinomycetes isolated from
marine samples are merely of terrestrial origin, notwithstanding the evidence that actinomycetes can be recovered
from sea and deep-ocean sediments, and that marinederived actinomycetes can be metabolically active (Weyland,
1969; Helmke & Weyland, 1984; Moran et al., 1995) and
physiologically adapted to growth in seawater (Jensen &
Fenical, 1994). Until recently, it has been suggested that
some members of the genera Rhodococcus, Dietzia, Streptomyces and Salinospora are indigenous actinomycetes in
marine ecological systems, according to their optimal
growth ability in in situ conditions including salinity,
temperature, pressure and nutrient concentration (Bull
et al., 2005). It has been reported that marine-derived
species affiliated to the family Micromonosporaceae form
glistening colonies that are purple or pale-to-bright orange
on the isolation medium with species specificity (Magarvey
et al., 2004). Furthermore, several groups of Actinobacteria,
including species from the genera Micrococcus and Corynebacterium, can synthesize cyclic and acyclic C45 and C50
carotenoids (Goodwin, 1980). In this study, 37 pigmented
representatives of the genera Rhodococcus, Dietzia, Micrococcus and Kocuria were obtained from our sampling sites,
with the majority (91.9%) from the Yangtze River Estuary
and coastal waters of Chinese marginal seas (Table 2).
Most isolates of Firmicutes were obtained from the
Yangtze River Estuary and clustered into class Bacilli. Few
publications are devoted to the study of Bacillus species in
marine environments, and until recently few Bacillus species
had been obtained from marine environments (Garabito
et al., 1997; Ivanova et al., 1999; Zhuang et al., 2003). Among
the numerous Bacillus species, only species of B. badius,
B. subtilis, B. cereus, B. lichenifirmis, B. firmus, B. pumilus,
B. mycoides, and B. lentus have been detected from marine
environments, including marine-derived species, such as
B. marinus, B. dipsosauri (Ivanova et al., 1999) and
B. salexigens (Garabito et al., 1997). Furthermore, marineoriginated species have been reported to produce unusual
metabolites, different from the species of terrestrial origin
(Zhuang et al., 2003). Here, 62 pigmented Bacilli-related
isolates were obtained and grouped into three genera,
Bacillus, Planococcus and Exiguobacterium, in the phylogenetic tree (Fig. 3d), while previous studies suggested that
marine Bacillus rarely showed pigmented forms, despite
several exceptional cases (Pane et al., 1996; Siefert et al.,
2000).
Bacteroidetes (previously called the Cytophaga–Flavobacteria–Bacteroides group) is a newly established phylum,
FEMS Microbiol Ecol 57 (2006) 92–105
103
Diversity and distribution of pigmented bacteria
including three classes: Bacteroidetes, Flavobacteria and
Sphingobacteria (Garrity et al., 2002; Kirchman, 2002).
Bacteroidetes are abundant in aquatic habitats when assessed
by fluorescent in situ hybridization and in 16S rRNA gene
libraries (O’Sullivan et al., 2004). The ecological significance
of Bacteroidetes bacteria has been brought to light because of
their proficiency in degrading various biopolymers such as
cellulose, chitin, and pectin (Kirchman, 2002). Colonies of
many Bacteroidetes bacteria exhibit yellow, orange, pink or
red pigmentation as a result of the flexirubin-type pigments
found in these bacteria (Kirchman, 2002). In this study,
most Bacteroidetes bacteria, with broad genetic diversity,
were obtained from the South China Sea, and no Bacteroidetes isolates were found in the Yangtze River Estuary.
Thus there is a discrepancy between our investigations and
previous studies, in which Bacteroidetes bacteria were commonly found in estuaries as a result of their ability to
catabolize riverine dissolved organic matter (Kisand et al.,
2002). It is proposed that, in the Yangtze River Estuary,
abundant Firmicutes and Actinobacteria bacteria are more
competitive in degrading organic matter or in growing on
agar plate than Bacteroidetes bacteria, which was revealed by
a previous study (Sekiguchi et al., 2002).
In conclusion, our initial work on the abundance distribution and the wide genetic diversity of PHB has shed
light on the potential ecological functions of PHB in marine
environments as a result of their expression of pigments.
Questions also arise regarding the speciation and roles of
PHB in the oceans. For example, compared with nonpigmented heterotrophic bacteria, what do PHB contribute to
the marine ecosystem, especially in terms of light absorption
in visible spectra, besides the well-known functions of their
pigments as antioxidants, light protection, and membrane
stabilizers?
Acknowledgements
We thank Dr Yao Zhang and Yong Ma for their assistance in
sampling, and Professors Senjie Lin, Kunming Xu and Dr
Serif Basoglu for their efforts in revising the manuscript.
This work was supported by the projects NSFC40232021,
NSFC 40576063, G2000078500, MOST2003, DF000040,
2001CB409700 and the MOE key project.
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