RESEARCH LETTER
Vibrio azureus emits blue-shifted light via an accessory blue
fluorescent protein
Susumu Yoshizawa1, Hajime Karatani2, Minoru Wada3 & Kazuhiro Kogure1
1
Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan; 2Department of Biomolecular Engineering, Graduate School
of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan; and 3Graduate School of Science and Technology, Nagasaki University,
Nagasaki, Japan
Correspondence: Susumu Yoshizawa,
Atmosphere and Ocean Research Institute,
The University of Tokyo, 5-1-5 Kashiwanoha,
Kashiwa, Chiba 277-8564, Japan.
Tel./fax: +81 4 7136 6419; e-mail:
[email protected]
Received 24 November 2011; revised 8
January 2012; accepted 9 January 2012.
Final version published online 10 February
2012.
DOI: 10.1111/j.1574-6968.2012.02507.x
MICROBIOLOGY LETTERS
Editor: Aharon Oren
Keywords
bioluminescence; luminous bacteria;
fluorescent protein; light emission; multilocus
sequence analysis.
Abstract
Luminous marine bacteria usually emit bluish-green light with a peak emission
wavelength (kmax) at about 490 nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein
(lumazine protein: LumP) that causes a blue shift, from kmax 490 to
kmax 476 nm. However, the incidence of blue-shifted light emission or the
presence of accessory fluorescent proteins in bacteria of the genus Vibrio has
never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio
azureus emit a blue-shifted light with a peak at approximately 472 nm, whereas
other Vibrio strains emit light with a peak at around 482 nm. Therefore, we
investigated the mechanism underlying this blue shift in V. azureus NBRC
104587T. Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that
this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light
emission spectrum of the strain. This result strongly suggested that VA-BFP
was responsible for the blue-shifted light emission of V. azureus.
Introduction
Luminous bacteria occur ubiquitously in marine environments and have been isolated from seawater, sediment,
detritus, and light-emitting organs of marine animals
(Reichelt & Baumann, 1973; Ramesh et al., 1990; Nealson
& Hastings, 1991; Dunlap & Kita-Tsukamoto, 2006). To
date, 23 species of luminous marine bacteria have been
identified, consisting of 11 Vibrio species, four Aliivibrio
species, six Photobacterium species, and two Shewanella
species (Gomez-Gil et al., 2004; Dunlap & Kita-Tsukamoto, 2006; Ast et al., 2007; Urbanczyk et al., 2007, 2008;
Yoshizawa et al., 2009a, b, 2010a, b, in press). Luminous
bacteria use bacterial luciferase to produce a bluish-green
light. The luciferase catalyzes the oxidation of reduced
riboflavin-5′-phosphate (FMNH2) with a long-chain aliphatic aldehyde and molecular oxygen, and the peak light
emission generally occurs around 490 nm (Hastings &
FEMS Microbiol Lett 329 (2012) 61–68
Nealson, 1977). This reaction is called luciferase–luciferin
reaction, and bacterial luciferase is composed of alpha
and beta subunits encoded by luxA and luxB, respectively
(Engebrecht et al., 1983).
Modulation of the light emission spectrum is often
observed among luminous organisms, such as Aequorea
victoria (Shimomura et al., 1962), and has been observed
in three species of luminous bacteria (Photobacterium
phosphoreum, Photobacterium leiognathi, and Aliivibrio
sifiae strain Y1 [formerly known as Vibrio fischeri strain
Y1]). The mechanism of this phenomenon was initially
characterized in P. phosphoreum strain A-13 (Gast &
Lee, 1978). The maximal emission wavelength (kmax 476 nm) of this strain is blue-shifted in comparison with
that of purified luciferase (kmax 490 nm). Gast & Lee
(1978) showed that this blue shift was caused by the
involvement of an accessory blue fluorescent protein, of
which the fluorescent spectrum was identical to the
ª 2012 Federation of European Microbiological Societies
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62
S. Yoshizawa et al.
in vivo light emission spectrum. This protein was also
found in P. leiognathi (O’Kane et al., 1985) and is now
called lumazine protein (LumP). LumP has 6,7-dimethyl8-(1′-D-ribityl) lumazine as its chromophore (Koka &
Lee, 1979). In another case, an accessory yellow fluorescent protein (YFP) was discovered in the yellow-lightemitting V. fischeri strain Y1 (Daubner et al., 1987),
which has been recently reclassified as A. sifiae (Ast et al.,
2009; Yoshizawa et al., 2010b). YFP modulates the light
emission wavelength of bacterial luciferase to yellow
(kmax 540 nm). These proteins are involved in the
luciferase reaction, and it is generally accepted that the
peak emission wavelengths of the light emission spectra
are shifted to shorter or longer wavelengths that correspond to the spectra of these fluorescent proteins (Gast &
Lee, 1978; Small et al., 1980; Karatani et al., 1992). There
are, however, no reports of an accessory fluorescent protein in bacteria of the genus Vibrio.
The aim of this study was to explore luminous bacteria
with modulated light emission in the genus Vibrio and to
see whether these bacterial strains carry an accessory fluorescent protein. We performed detailed analyses of the
light emission spectra and the luxA gene sequences in 16
strains of four luminous Vibrio species (Vibrio harveyi,
Vibrio campbellii, Vibrio azureus, and Vibrio jasicida).
Multilocus sequence analysis (MLSA) was used for bacterial identification. Furthermore, the protein involved in
the shift was purified and subjected to spectral base characterization in vitro. As a result, we obtained a new fluorescent protein responsible for the blue-shifted light
emission of V. azureus.
Materials and methods
Bacterial strains used in this study
We used 16 luminous strains of genus Vibrio (Table 1).
Bacterial strains newly reported in this study were isolated from seawater samples from Sagami Bay (35°00′N,
139°20′E), the Pacific equatorial zone, and Aburatsubo
Inlet (35°09′N, 139°36′E). We used Niskin bottles to
collect seawater samples in Sagami Bay during the
KT-05-16 cruise of R/V Tansei Maru (Atmosphere and
Ocean Research Institute, University of Tokyo [AORIUT] and Japan Agency for Marine-Earth Science and
Technology [JAMSTEC]) and in the Pacific equatorial
zone during the KH-04-5 cruise of R/V Hakuho Maru
(AORIUT and JAMSTEC). Surface seawater samples
were collected at Aburatsubo Inlet by using 50-mL
Corning tubes (Sigma-Aldrich Japan, Tokyo, Japan)
(Table 1). The method used for isolating luminous colonies was as described previously (Yoshizawa et al.,
2009b).
Bacterial identification by 16S rRNA gene
sequence analysis, and phylogenetic analyses
based on the luxA gene and MLSA
A Bio-Rad AquaPure Genomic DNA Kit (Bio-Rad Laboratories, Hercules, CA) was used to extract genomic DNA
from 1 mL overnight cultures of strains grown in ZoBell
broth. The 16S rRNA gene was amplified with bacterial
universal primers (Lane, 1991). Other primers designed
Table 1. Bacterial strains used in this study for measuring emission spectra
Emission spectra*
Sampling point or source
†
Species
Strain
kmax (nm)
FWHM (nm)
Latitude
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
LC1-863
LC1-870
LC1-900
LC1-943
LC1-953
ABR2-3
NBRC 15634T
LC1-984
LC1-989
NBRC 104587T
LC2-025
LC2-045
LC2-050
ABR3-1
LC2-032
LC2-055
481
480
484
484
479
481
480
471
480
472
469
471
471
482
484
482
86
86
87
84
84
84
85
65
81
67
66
68
66
84
88
85
5°00′S
5°00′S
5°00′S
5°00′N
5°00′N
35°09′N
campbellii
campbellii
campbellii
campbellii
campbellii
harveyi
harveyi
azureus
azureus
azureus
azureus
azureus
azureus
jasicida
jasicida
jasicida
Longitude
Depth (m)
170°00′W
100
170°00′W
100
170°00′W
200
170°00′W
5
170°00′W
75
139°36′E
0
Dead plankton (Talorchestia sp.)
35°00′N
139°20′E
30
35°00′N
139°20′E
50
32°30′N
138°00′E
200
35°00′N
139°20′E
30
35°00′N
139°20′E
0
35°00′N
139°20′E
20
35°09′N
139°36′E
0
35°00′N
139°20′E
30
35°00′N
139°20′E
30
*Culture condition: 20 °C, 24–48-h incubation.
Full width at half maximum.
†
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FEMS Microbiol Lett 329 (2012) 61–68
63
Blue-shifted light-emitting Vibrio
and used for amplification of the luxA gene, which encodes
the alpha subunit of luciferase, were Vch LuxA-F (5′-GAT
CAAATGTCAAAAGGACG-3′) and Vch LuxA-R (5′-CC
GTTTGCTTCAAAACCACA-3′). Genes encoding uridylate
kinase (pyrH), a cell division protein (ftsZ), and a rodshaped protein (mreB) were used for MLSA (Thompson
et al., 2007). PCR primers for the three genetic loci and
reaction conditions were used in accordance with the
method of Sawabe et al. (2007). TaKaRa EX Taq polymerase (TaKaRa Bio, Shiga, Japan) was used to amplify the
genes. An ABI Prism 3100 Genetic Analyzer (Applied
Biosystems, Foster City, CA) was used for sequencing.
Multiple alignments of the sequences were performed with
CLUSTAL W (version 1.6) (Thompson et al., 1994). Distances
were calculated by using the Kimura 2-parameter model
(Kimura, 1980). Clustering based on the neighbor-joining
method (Saitou & Nei, 1987) was determined using bootstrap values based on 1000 replications (Felsenstein, 1985).
Sequence data used for other Vibrio species were from the
online electronic taxonomic scheme for Vibrios (http://
www.taxvibrio.lncc.br) and the GenBank database.
Measurement of light emission spectra
In vivo light emission spectra of luminous strains were
measured after incubation at 20 °C for 24–48 h on ZoBell
2216E agar medium. Fluorescence (emission and excitation) and light emission spectra (in vivo and in vitro)
were measured with a Shimadzu Model RF-5300PC spectrofluorophotometer (Shimadzu, Kyoto, Japan). Light
emission spectra were measured more than twice with the
excitation lamp off. For all measurements, the wavelength
scan rate was 50 nm s 1.
Cell culture and protein isolation
Cells of V. azureus strain NBRC 104587T were grown in
ZoBell broth at 27 °C. The cells were harvested in the
second half of the exponential phase. Subsequent procedures were carried out at 4 °C. The cells of NBRC
104587T were osmotically lysed in 10 mM Na/K phosphate lysis buffer containing 10 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT)
(pH 7.0). The lysate was centrifuged for 60 min at
10 000 g, and the supernatant was collected. Proteins
were fractionated by the addition of solid ammonium
sulfate to the cell lysate. The proteins that precipitated at
between 40% and 80% (NH4)2SO4 saturation were collected by centrifugation for 60 min at 10 000 g. The protein precipitates were then dissolved in 10 mM Na/K
phosphate buffer (containing 0.1 mM EDTA, 1 mM DTT
[pH 7.0]; 1 mL g 1 of precipitate) and dialyzed three
times with fresh dialysis buffer.
FEMS Microbiol Lett 329 (2012) 61–68
Purification of blue fluorescent protein and
luciferase
Proteins of interest were isolated from the dialyzed
V. azureus NBRC 104587T cell lysate by means of a series
of chromatographic steps in accordance with the protocol
described by Karatani et al. (1992).
Purification of Vibrio azureus NBRC 104587T
flavin reductase
The flavin reductase – pooled fractions from the DEAE
(diethylaminoethyl cellulose) column – was concentrated
by ultrafiltration and then loaded onto a Sephacryl S-200
HR column (bed volume, 37 mL; height, 65 cm; diameter,
15 mm). Elution proceeded at a flow rate of 11 mL h 1.
Flavin reductase activity was determined according to the
method described by Jablonski & DeLuca (1977). Protein
concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad) with bovine serum albumin as a
standard.
In vitro luciferase assays
Luciferase activity was measured by using a nonturnover
assay at 20 °C (Hastings et al., 1978). In brief, 1 mL of
50 lM FMNH2, prepared from FMN on Pt-asbestos, was
quickly injected into a reaction mixture containing 20 lL
of 0.1% (w/v) dodecanal emulsified in H2O, 100 lL of
100 mM Na/K phosphate buffer (pH 7.0), and 20 lL of
luciferase. To measure the in vitro light emission spectrum, a reaction was initiated by quick injection of
190 lL of 100 lM nicotinamide adenine dinucleotide in
reduced form (NADH) in a reaction mixture containing
20 lL of luciferase (20 lM), 20 lL of flavin reductase
(2 lM), 20 lL of 0.1% (w/v) aliphatic aldehyde (dodecanal), 50 lL of FMN (100 lM), and 100 lL of 100 mM
Na/K phosphate buffer.
Results and discussion
Bacterial strains used in this study
Except for V. harveyi NBRC 15634T and V. azureus
NBRC 104587T, the luminous strains used in this study
were not type strains (see Table 1). We used the 16S
rRNA gene and three house-keeping genes for identification of these strains, because phylogenetic analysis on the
basis of only 16S rRNA gene data is not adequate for the
identification of bacteria in the genus Vibrio (Thompson
et al., 2005). Phylogenetic analysis based only on 16S
rRNA gene sequence data (Supporting Information,
Fig. S1) suggested that all strains used in this study were
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Published by Blackwell Publishing Ltd. All rights reserved
64
S. Yoshizawa et al.
included in the Harveyi clade (Sawabe et al., 2007). For
this reason, these strains were identified by MLSA using
three genetic loci (pyrH, ftsZ, and mreB: total length
1274 bp). The phylogenetic tree constructed from MLSA
is shown in Fig. 1. The classification results and detailed
information about the sources of luminous strains used
in this study are described in Table 1.
(FWHM) is listed in Table 1. The spectral distributions
of light emitted by V. campbellii, V. harveyi, and V. jasicida are almost identical (Fig. 2: upper panel). These
spectra show a nearly symmetrical broad distribution
about a peak emission at a wavelength of approximately
482 nm (FWHM values are around 85 nm). On the
other hand, all strains of V. azureus, except for LC1-989,
produced light with a peak emission at approximately
472 nm in a narrow spectral band as indicated by a
FWHM value of 66 nm (Fig. 2: lower panel). The emission spectrum of LC1-989 has a maximum wavelength
of 480 nm and a broad shape (FWHM value of 81 nm)
and is similar to the spectra of V. campbellii, V. harveyi,
and V. jasicida. Widder and her colleagues reported that
the light emission spectra of V. harveyi have the peak at
483 and 488 nm (FWMH values are 93 and 96 nm,
respectively) (Widder et al., 1983). Another paper mentions that the emission peak of V. harveyi is at around
490 nm (Herring, 1983). To our knowledge, a light
Light emission spectral analysis of luminous
Vibrio species
To examine the light emission spectra of these strains,
we used luminous colonies incubated at 20 °C for 24–
48 h. ZoBell 2216E agar medium was used for cultivation because most luminous strains in the genus
Vibrio emit light that is too dim for measurement when
cultivated in broth media. The emission spectrum of
each species is shown in Fig. 2, and the wavelength of
maximum emission and full width at half maximum
pyrH
ftsZ
LC1-863
mreB
93
LC1-943
LC1-870
Total length 1274 bp
LC1-953
70
V. campbellii CAIM 155
100
58
Vibrio campbellii
V. campbellii CAIM 372
V. campbellii LMG 11216T
95
LC1-900
73
V. harveyi NBRC 15634T
V. harveyi 818ODDZ10
100
94
V. harveyi CAIM 107
76
67
Vibrio harveyi
ABR2-3
100
V. rotiferianus 1975
V. rotiferianus LMG 21460T
72
ABR3-1
54
LC2-032
100
97
Vibrio jasicida
LC2-055
93
97
96
V. jasicida TCFB 0772 T
V. jasicida TCFB 1977
V. alginolyticus LMG 4409T
V. parahaemolyticus LMG 2850T
V. natriegens LMG 10935T
75
LC2-050
LC2-045
100
LC2-025
NBRC 104587T
100
Vibrio azureus
LC1-989
0.02
70
LC1-984
V. vulnificus LMG 13545T
V. splendidus LMG 19031T
Aliivibrio fischeri ATCC 7744T
Photobacterium leiognathi LMG 4228T
Fig. 1. Phylogenetic tree based on the neighbor-joining method, using concatenated gene sequences of the three loci (1274 bp). Bold characters
indicate the luminous strains used in this study. Bootstrap values are expressed as percentages of 1000 replications; only values > 50% are
shown. Photobacterium leiognathi was used as an outgroup. Scale bar, 2% estimated sequence divergence.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Lett 329 (2012) 61–68
65
Blue-shifted light-emitting Vibrio
Normalized light Intensity
1.2
1
Vibrio campbellii
Vibrio harveyi
0.8
Vibrio jasicida
0.6
0.4
0.2
0
Normalized light Intensity
bution. In the case of Photobacterium, the spectrum of
blue-shifted light emission induced by LumP (kmax 476 nm) also has an asymmetric shape and is narrower
than the light emission produced by purified luciferase
(Gast et al., 1978). It is, therefore, most likely that the
light emission with the peak at 472 nm produced by
V. azureus was a result of the luciferase–luciferin reaction interacting with an accessory protein.
ca. 482 nm
Phylogenetic analyses using the luxA gene
ca. 472 nm
480 nm
1
To examine whether the primary structure of luciferase
could affect the light emission spectra, we determined the
luxA gene sequences of the strains and analyzed these
data. The phylogenetic tree based on the amino acid
sequence data of luxA showed that the strains were clustered by species (Fig. 3). It has been reported previously
that the luxA gene is useful in taxonomic and phylogenetic analyses of luminous bacteria (Haygood & Distel,
1993; Dunlap & Ast, 2005; Wada et al., 2006), and our
analyses based on the luxA gene and MLSA also support
these reports. However, this tree could not discriminate
LC1-989 from the other V. azureus strains, because the
sequence data of LC1-989 shares 100% sequence identity
with that of V. azureus NBRC 104587T. It is clear from
this result that the light emissions peaking at 472 nm
were not owing to any structural differences in luciferase,
but were most likely due to the presence of other components, such as accessory fluorescent proteins. The GenBank accession numbers of sequences obtained in this
study are shown in Table S1.
Vibrio azureus
Vibrio azureus
strain LC1-989
0.8
0.6
0.4
0.2
0
400
450
500
Wavelength (nm)
550
600
Fig. 2. Spectral distributions of light emitted by luminous bacteria.
Vibrio campbellii, n = 5; Vibrio harveyi, n = 2; Vibrio jasicida, n = 3;
Vibrio azureus, n = 5. Vertical line indicates the position of 480 nm.
emission peak at a wavelength shorter than 480 nm has
not been previously reported for the genus Vibrio. In
addition, the shape of the spectrum produced by
V. azureus tended to deviate from a Gaussian-like distri-
LC1-953 (AB428944)
Vibrio campbellii (EF394780)
LC1-943 (AB428943)
61
LC1-900 (AB428942)
Vibrio campbellii
LC1-870 (AB428941)
100
LC1-863 (AB428940)
V. harveyi NBRC 15634T (DQ436496)
99
63
ABR2-3 (AB428939)
Vibrio harveyi
LC2-032 (AB428953)
100
74
ABR3-1 (AB428954)
Vibrio jasicida
LC2-055 (AB428955)
LC1-984 (AB428945)
LC1-989 (AB428958)
NBRC 104587T (AB428946)
100
LC2-025 (AB428947)
Vibrio azureus
LC2-045 (AB428948)
LC2-050 (AB428949)
Aliivibrio fischeri ATCC 7744T (AY341062)
0.05
Fig. 3. Phylogenetic tree derived from neighbor-joining analysis of partial luxA amino acid sequences. Boldface type indicates blue-shifted strains.
Numbers above nodes represent bootstrap confidence values obtained from 1000 resamplings. Aliivibrio fischeri was used as an outgroup. Scale
bar, 5% estimated sequence divergence.
FEMS Microbiol Lett 329 (2012) 61–68
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
66
S. Yoshizawa et al.
Purification of blue fluorescent protein
Spectral properties of VA-BFP
We compared the in vivo light emission spectrum of
V. azureus NBRC 104587T with the in vitro light emission
spectrum from purified luciferase at 20 °C (Fig. 4). The
peak wavelengths of these two light emission spectra differed by about 16 nm, and the in vivo light emission
spectrum was narrower than the in vitro spectrum with
the FWHM value of the in vivo light emission spectrum
approximately 65 nm and that of the in vitro luciferase
reaction approximately 87 nm. The fluorescence emission
maximum of the isolated VA-BFP was in good agreement
with the in vivo light emission maximum (kmax 472 nm) of V. azureus NBRC 104587T (Fig. 4). From
these analyses, we concluded that VA-BFP isolated from
V. azureus NBRC 104587T is the substance causing the
blue-shifted light emission. In addition, the spectral
distribution of the light emitted by V. azureus NBRC
104587T is very similar to the spectrum of light emitted
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Normalized light intensity
From the results described above, we assumed that
V. azureus, except for LC1-989, would carry an accessory
blue fluorescent protein that modulates the light emission. We chose to examine NBRC 104587T, whose light
emission spectrum peaks at 472 nm, for further biochemical analysis of bacterial intracellular proteins.
The extracted proteins were initially purified by using
ion-exchange liquid chromatography (DEAE Sepharose).
During the first gradient step (10–250 mM), a fluorescent
component was eluted along with flavin reductase (Fig.
S2). The fluorescent component had a fluorescence maximum wavelength of 470 nm and is therefore referred to
as F470 in this paragraph. Luciferase was eluted in the
second gradient step (250–1500 mM). Similar chromatographic behavior was observed for the accessory fluorescent protein produced by A. sifiae strain Y1 (Karatani
et al., 1992; Karatani & Hastings, 1993). F470 was subjected to gel filtration chromatography, and SDS-PAGE
analysis of the eluate indicated that the molecular size of
F470 was approximately 23 kDa (Fig. S3, lane 7). On the
basis of the A280/A414 value (= 2.3), F470 was determined
to be pure enough for characterization (O’Kane et al.,
1985), with only a negligible level of contaminants
remaining. We termed the purified blue fluorescent protein component (F470) VA-BFP. Luciferase was further
purified by means of gel filtration chromatography
and affinity chromatography (detailed information is
described in Materials and methods). The upper and
lower bands of purified luciferase proteins (Fig. S3, lane
5) represent luciferase alpha and beta subunits, respectively.
1.2
VA-BFP
fluorescent spectrum
In vivo
In vitro
1
0.8
0.6
0.4
0.2
0
400
450
500
550
600
Wavelength (nm)
Fig. 4. Fluorescence emission spectrum of 20 mM VA-BFP (dotted
line) in 100 mM Na/K phosphate buffer (pH 7.0), excitation at
415 nm. In vivo Vibrio azureus NBRC 104587T light emission
spectrum (solid line). In vitro light emission spectrum (dashed line)
was measured in the reactions initiated by rapid injection of 190 lL
of NADH (100 lM) in a reaction mixture containing 20 lL of
luciferase (20 lM), 20 lL of flavin reductase (2 lM), 20 lL of 0.1%
(w/v) dodecanal emulsified in water, 50 lL of FMN (100 lM), and
100 lL of 100 lM Na/K phosphate buffer. All spectra were measured
at 20 °C.
by the genus Photobacterium, although the maximal wavelength is approximately 5 nm shorter. This indicates that
VA-BFP carries the 6,7-dimethyl-8-(1′-D-ribityl) lumazine
chromophore, as identified in LumP (Koka & Lee, 1979).
Conclusion
Vibrio harveyi has been known as a luminous bacterium
since the 1930s (Johnson & Shunk, 1936) and has come
to be luminous representative of the genus Vibrio; therefore, almost all investigations on the genus Vibrio have
been conducted on this representative species. However, a
modulated light emission spectrum induced by an accessory fluorescent protein had never been observed in this
group. In this paper, we examined the light emission
spectra of luminous strains in the genus Vibrio, focusing
on the involvement of an accessory fluorescent protein.
Because recent research has found many luminous strains
in species related to V. harveyi (Gomez-Gil et al., 2004;
Yoshizawa et al., 2009b), we analyzed the light emission
spectra of not only V. harveyi but also other Vibrio species.
Light emission spectral analysis revealed two types of
light emission spectrum: symmetrical light emission spectra having a broad shape and a peak at approximately
482 nm and asymmetrical (blue-shifted) light emission
spectra of a narrower shape with a peak at approximately
472 nm. Moreover, we succeeded in purifying VA-BFP
from a strain of V. azureus with blue-shifted light emission. This is the first report of blue-shifted light emission
FEMS Microbiol Lett 329 (2012) 61–68
Blue-shifted light-emitting Vibrio
and an accessory blue fluorescent protein among luminous bacteria of the genus Vibrio.
Acknowledgements
We are grateful to the officers and crew of the R/V Tansei
Maru and R/V Hakuho Maru for their assistance and support in sample collection. We also thank Kumiko KitaTsukamoto for the technical support and Nami Uchiyama
for bacterial isolation. This study was supported in part
by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (No. 17580156; No.
17310127) and by a Sasakawa Scientific Research Grant
from the Japan Science Society.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Phylogenetic tree based on the neighbor-joining
method, using partial 16S rRNA gene sequences.
Fig. S2. DEAE column elution pattern for the dialyzed
ammonium sulfate–precipitated fraction of Vibrio azureus
NBRC 104587T cell lysate.
Fig. S3. SDS-PAGE patterns of luciferase and blue fluorescent protein at each stage in the purification.
Table S1. The GenBank accession numbers of sequences
obtained in this study.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
FEMS Microbiol Lett 329 (2012) 61–68