Stimulated biosynthesis of flavins in
Photobacterium phosphoreum
IFO 13896 and the presence of complete
rib
operons in two species
of luminous bacteria
Sabu Kasai and Takumi Sumimoto
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Japan
Photobacterium phosphoreum IFO 13896 emits light strongly
when cultured in medium containing 3% NaCl, but only
weakly in medium containing 1% NaCl. It is known that
dim or dark mutants appear frequently and spontaneously
from this parent strain. To confirm that riboflavin biosyn-
thesis is stimulated when the lux operon is active, the amount
of light emitted and flavins synthesized under strongly or
weakly light emitting conditions was determined. In com-
parison with the parent strain cultured in 3% NaCl, the same
strain cultured in 1% NaCl emitted 1/36 the light and pro-
duced 1/4 the flavins, while three dim or dark mutants, M1,
M2andM3culturedin3%NaCl,emittedalmostnolight,
1/58 the light and 1/10 the light and produced 1/8, 1/5 and
1/3 the amount of flavins, respectively. From these results,
we deduced that the genes for riboflavin synthesis, rib genes,
are organized in an operon in this strain. In P. phosphoreum
NCMB 844, it has been reported that a rib gene cluster is
present just downstream of the lux operon. However, among
rib genes, the gene for pyrimidine deaminase/pyrimidine
reductase, ribD, was not found in this cluster. Because a
complete rib operon seems to be necessary for efficient
regulation at the transcriptional level, we expected ribD to be
present downstream of this cluster and sequenced this

and 3,4-dihydroxy-2-butanone 4-phosphate synthase are
not fused and they are designated ribA and ribB,
respectively [2]. The genes corresponding to ribG, ribB
and ribH of B. subtilis are designated ribD, ribC and ribE,
in E. coli, respectively, and if a gene corresponding to
ribA of B. subtilis is present in other species, it is called
ribBA. To avoid confusion, we have used the E. coli
notational system for rib genes in this report.
About 20 years ago, we isolated roseoflavin-resistant
mutants from Gram-positive bacteria including B. subtilis,
and found that they acquire their antibiotic resistance
through the overproduction of riboflavin [3]. Later, it was
found that in B. subtilis, rib genes are organized in an
operon and the overproduction in mutants is caused by the
deregulation of this operon [1]. Now genome sequences of
many species of Gram-positive bacteria have been released
and in riboflavin autotrophs of these bacteria rib genes are
organized in an operon. In E. coli, however, such riboflavin-
overproducing mutants have not yet been identified because
rib genes are not organized in an operon but scattered
around the chromosome [1,4], and their expression is
constitutive [2]. Because in two genome-sequenced Gram-
negative bacteria, Haemophilus influenzae [5] and Helico-
bacter pylori [6], the rib genes are not organized in an
operon, it is believed that in Gram-negative bacteria the rib
genes are not organized in an operon and stimulated
synthesis of riboflavin does not occur.
Correspondence to S. Kasai, Department of Bioapplied Chemistry,
Faculty of Engineering, Osaka City University, Sugimoto 3-3-138,
Sumiyoshi-ku, Osaka 558–8585, Japan.

species are classified as Gram-negative bacteria. Accord-
ingly, it is quite possible that there is a complete rib operon
also in P. phosphoreum IFO 13896.
We attempted to confirm that P. phosphoreum
IFO 13896 synthesizes flavins at enhanced levels when
strongly emitting light and then to identify a complete rib
operon in this species. We expected ribD to be present just
downstream of the rib cluster reported by Lee et al. [7].
However, we could not find it although we found another
complete rib operon in a different region. Because rib
operons are duplicated in P. phosphoreum, albeit incom-
pletely, there are three rib genes of the same name in one
species. So, in this report, to distinguish among duplicated
genes, we give names with prime such as ribC¢, ribBA¢, ribE¢
and ribA¢, for genes in the rib gene cluster located just
downstream of the lux operon, and names without prime
such as ribD, ribC, ribBA and ribE, for those in the newly
found complete rib operon.
MATERIALS AND METHODS
Bacteria
P. phosphoreum IFO 13896 is almost the same strain as
NCMB 844 [10] and emits much light. The strain is
maintained in our laboratory and the Institute for Fermen-
tation, Osaka (IFO). Dark or dim mutants, M1, M2 and
M3, appeared spontaneously on agar plates with cultures of
the parent strain, P. phosphoreum IFO 13896, and were
isolated.
Growth and bioluminescence curves
A diluted medium was used to obtain growth curves
ensuring that the maximum turbidity of the liquid culture

the parent strain and three mutants were grown until a
turbidity of  7 in the case of the parent strain cultured in
3% NaCl medium and 10–13 in the other cases. For the
parent strain grown in 3% NaCl, cells in 3 mL culture,
while in other cases cells in 4.5 mL, were collected by
centrifugation at 16 000 g for 5 min. The supernatant was
discarded and any remaining was removed by using filter
paper. Each cell pellet was suspended in 1.5 mL distilled
water using a vortex mixer. After heating at 95 °Cfor
5 min, the suspension was sonicated for 5 min. After a
second heating at 95 °C for 3 min, cell debris were collected
by centrifugation at 16 000 g for 5 min. Aliquots (1 mL) of
each supernatant were measured. Total flavins were deter-
mined using a lumiflavin fluorescence method [11] with
slight modification: 1 mL each sample was added to 1 mL
1
M
NaOH in a 10-mL test tube and photolysed under a
fluorescent lamp for 10 min The photoproduct, lumiflavin,
was extracted with 3 mL chloroform and quantified fluor-
ometrically. Total flavins in the cell pellet collected from
1 mL of the culture giving a turbidity of 1, in the respective
sample, were calculated.
Because P-flavin, 6-(3¢-myristic acid)-FMN and 6-(4¢-
myristic acid)-FMN, are present in extracts of P. phos-
phoreum cells [12] we examined whether the lumiflavin
fluorescence method can be used to quantify P-flavin in a
solution of FP
390
, Lux F, which binds this flavin [10,13–15].

respective template for sequencing, two primers, either
nondegenerate or degenerate, were designed and then a
DNA sequence was amplified using Ready-To-Go PCR
beads (Amersham Biosciences) with a temperature control
program of 20 s at 94 °C, 20 s at 50 °C, and 2 min at 72 °C
for 40 cycles. The amplified DNA sequences were purified
by means of agarose gel electrophoresis and then gel
extraction kits (QIAGEN).
Nucleotide sequences were determined using a BigDye
Terminator Cycle Sequencing Ready Reaction Kit and an
ABI PRISM 310 genetic analyser (Applied Biosystems) at
the Graduate School of Science, Osaka City University,
according to the manufacturer’s protocol. In this study, we
first amplified the DNA to be sequenced as above and then
directly sequenced it using the primers for amplification
without cloning [17]. The protocol for SUGDAT, Sequen-
cing Using Genomic DNA As a Template, was reported
previously [17] and it worked well also in the sequencing of
the P. phosphoreum genomic DNA. When SUGDAT was
repeated, the average length of determined sequences was
 300 bp because nonerroneous and non-AT-rich sequences
were necessary to design primers for the subsequent
SUGDAT. In this study, three DNA sequences, the total
lengths of which were 1995, 5616 and 5232 bp, were
sequenced by SUGDAT and a PCR-based method using
degenerate primers as described below. To exclude reading
errors, DNA sequences of appropriate lengths (400–500 bp)
where neighbouring sequences overlapped were amplified
by PCR as described above and sequenced once again on
both strands.

PCR product. Further, a conserved amino acid sequence
in RibE, NKGAEAA, was found by comparing RibE
sequences from V. cholerae (AE004298), P. phosphoreum
(RibE¢) (L11391), P. leiognathi (M90094), Ps. aeruginosa
(AE004821) and E. coli (X64395), and a degenerate
reverse primer, 5¢-GCNGCYTCIGCNCCYTTRTT-3¢ was
designed. Using this primer and a forward one, a 1267-bp
sequence was amplified and sequenced. The undetermined
part of ribE and an 807-bp sequence further downstream
were sequenced by three rounds of SUGDAT.
The sequencing of the rib operon with both flanking
regions in V. fischeri ATCC 7744 (5232 bp) is outlined in
Fig. 1C. Partial of ribD was amplified using the same
primers as above, and sequenced. A 1221-bp upstream
sequence was determined by six rounds of SUGDAT. A
1554-bp and a 1232-bp sequence were amplified using the
two degenerate primers above, and two nondegenerate
primers. Further, a 520-bp sequence was amplified using a
degenerate primer, 5¢-ACNCCRTTNACRAAYTTRTG-3¢
and a nondegenerate primer. The former was designed
according to a conserved amino acid sequence in NusB,
HKFVNGV, which was found by comparing NusB
sequences from P. phosphoreum as determined above,
V. cholerae (AE004298), E. coli (X64395) and Ps. aerugi-
nosa (AE004821). The last 262-bp sequence was determined
by one round of SUGDAT.
The three nucleotide sequences of hcp (1995 bp),
the complete rib operon with some genes (5616 bp) of
Fig. 1. Clusters of riboflavin biosynthetic genes in P. phosphoreum and
V. fis cheri. Open arrows indicate genes in these orientations. Solid

[18–20]. Meanwhile, dim or dark mutants appear sponta-
neously and frequently from Photobacterium species [21].
These poor light-emitting cells seemed to be useful as a
control to confirm that the biosynthesis of flavins is
stimulated in the cells strongly emitting light. Therefore,
we measured the amount of light and total amount of
flavins produced in the cells. Five growth curves of the
parent strain cultured in 3% and 1% NaCl, and M1, M2
andM3in3%NaClareshowninFig.2A.Growthrates
and maximum cell densities under the respective conditions
were not identical. In mutants cultured in 3% NaCl, growth
rates were almost the same; however, maximum cell density
was highest for M1 (designated 100%), 93% for M3, and
88% for M2. The growth rate of the parent strain cultured
in 1% and 3% NaCl, was 67% and 33% of that of the three
mutants, respectively. The maximum cell density of the
parent cells cultured in 1% and 3% NaCl was 82% and
73% of that of M1, respectively. Growth and biolumines-
cence curves of the parent strain cultured in 3% or 1%
NaCl and M1, M2 or M3 in 3% NaCl are shown in Fig. 2
(B1–B5). To estimate the amount of bioluminescence
emitted under each set of conditions, the respective biolu-
minescence curve was integrated. However, because maxi-
mum cell density varied with the conditions or strain, it was
necessary to normalize each value. Accordingly, we divided
the integrated value of bioluminescence by the maximum
cell density of the respective growth curve to estimate the
average bioluminescence emitted during growth causing an
increase in turbidity of 1 unit, which we designated as
specific bioluminescence. These values are shown in Table 1.

2.5-times more flavin than M1 cells, respectively. From
these results, we concluded that the biosynthesis of flavins is
regulated strictly in P. phosphoreum andthestraincanemit
much light because it can produce flavins on demand when
the expression of the lux operon is activated.
Identification of a gene for hybrid-cluster protein,
Hcp, in
P. phosphoreum
Biosynthesis is regulated by many mechanisms. Among
them the regulation of gene expression is important and for
efficiency genes are often organized in an operon in
prokaryotes. Because the biosynthesis of flavins is stimula-
ted vigorously on demand in P. phosphoreum as described
above, we speculated that rib genes are organized in an
operon in this species. Because a gene coding for pyrimidine
deaminase/pyrimidine reductase, ribD, is not present in the
rib cluster reported by Lee et al. [7], which is located just
downstream of the lux operon (Fig. 1A), we sequenced the
genomic DNA of P. phosphoreum IFO 13896 downstream
of the terminal region of ribA¢ asshowninFig.1A,
assuming that ribD may be present here. A 270-bp
nucleotide sequence was obtained in the first SUGDAT.
A partial sequence of ribD was not found in this sequence,
but a partial gene in the opposite orientation was found. We
compared the 48-amino acid sequence deduced from this
partial gene sequence with the sequences of proteins
collected in the DAD database using the
FASTA
program
at DDBJ and found it to be  60% identical to the

amino acids). The P. phosphoreum Hcp shows 62–40%
amino acid identity with the Hcp of these species. We
therefore concluded that hcp is present just downstream of
ribA¢ in P. phosphoreum.
Identification of another complete
rib
operon in
P. phosphoreum
IFO 13896 and analysis of its 5616-bp
sequence
The ribD gene was not found downstream of ribA¢ in
P. phosphoreum but the gene is surely present because
riboflavin cannot be synthesized without RibD. To find it,
we tried to amplify part of the gene by PCR using two
degenerate primers and obtained a DNA sequence of the
expected length (Fig. 1B). Because the amino acid sequence
deduced from this nucleotide sequence showed 56% identity
with that of RibD of V. cholerae (AE004298), we concluded
that we had amplified an expected partial ribD sequence.
After two rounds of SUGDAT downstream of the partial
ribD, we recognized that another ribC is present in
succession. Because this arrangement is generally found in
the rib operons of other species we expected ribBA to also be
present downstream of ribC. We therefore designed a
degenerate primer according to the conserved amino acid
sequence in RibBA, and amplified an 853-bp sequence. A
partial amino acid sequence deduced from the nucleotide
sequence showed 78% identity with that of RibBA of
V. cholerae, and we concluded that ribBA is present
downstream of ribC in P. phosphoreum. At this stage, we

in other species (Table 2). Although in E. coli, ribA and ribB
are separated, in P. phosphoreum they were fused as one
gene, ribBA, as in other species in which rib genes are
organized in an operon. The ribBA gene is separated from
ribE by 202 bp (Fig. 3), but a similar long spacing, 215 bp,
is also found in V. cholerae (AE004298). The homologies of
these five rib gene products with those of five different
species, along with RibC¢,RibBA¢ and RibE¢ of P. phos-
phoreum or P. leiognathi, and the number of amino acid
residues composing the respective protein are shown in
Table 2. From these data, we concluded that a complete rib
operon is present in P. phosphoreum, which explains why
this species can synthesize flavins at enhanced levels to
sustain a strong light emission. On the other hand, the
question of why a rib cluster, the lux–rib cluster, is present
downstream of the lux operon, arises. The respective rib
gene product, RibC, RibBA or RibE, in the newly found rib
operon showed the highest identity with the corresponding
gene product in the lux–rib operon of the same species,
RibC¢,RibBA¢ or RibE¢, among gene products from any
other species, as shown in Table 2. This evidence indicates
that the genes in the lux–rib cluster were not incorporated
into the genome of this species by horizontal transfer.
Because the lux–rib cluster does not contain ribD,this
incomplete operon would be unlikely to contribute mainly
to riboflavin biosynthesis and the cluster seems to be an
auxiliary operon. It was recently reported that a homolog-
ous rib cluster is present in a closely related species,
P. leiognathi [27]. In the lux–rib cluster, ribA¢ is present
along with ribBA¢ as shown in Fig. 1A. Fassbinder et al.

identity (%) in the sequences of five Rib proteins of P phosphoreum (P.p.) or V. fischeri (V.f.) with respect to one of the seven species and number of
amino acid residues composing the respective protein of each species (No. aa).
RibX RibD RibC RibBA RibE
P.p. V.f. No. aa P.p. V.f. No. aa P.p. V.f. No. aa P.p. V.f. No. aa P.p. V.f. No. aa
Number of amino acid residues 149 149 388 372 218 218 367 369 156 156
P. p vs. V. f 81 57 73 73 86
Species compared
P. phosphoreum (RibC¢, RibBA¢, RibE¢) – – – – 73 65 218 77 65 363 87 78 155
P. leiognathi (RibC¢, RibBA¢, RibE¢) – – – – 60 58 218 73 64 364 78 75 144
V. cholerae 84 84 156 57 62 367 62 69 217 70 81 369 82 91 173
E. coli 82 80 149 59 51 367 35 34 213 – – 68 67 156
Ps. aeruginosa 71 67 154 49 48 373 61 56 219 58 58 365 69 65 158
B. subtilis 48 47 152 41 44 361 38 40 215 42 44 398 52 53 154
Fig. 3. Nucleotide sequence of a newly found rib operon in P. phos-
phoreum with the deduced amino acid sequences of GlyA, RibX, RibD,
RibC, RibBA, RibE, NusB and ThiL. The nucleotides are numbered on
the left, and the amino acid residues are numbered on the right. The
location of each gene is indicated at the head of the gene. The asterisks
indicate the translational termination codons.
Ó FEBS 2002 Stimulated synthesis of flavins in P. phosphoreum (Eur. J. Biochem. 269) 5857
synthase (ribB) is one of the LuxR- and acylhomoserine
lactone-controlled nonlux genes, and also that the gene is
monocistronic and not a member of the rib operon [29]. This
poses the question of whether rib genes are organized in
an operon and the genes for 3,4-dihydroxy-2-butanone
4-phosphate synthase and GTP cyclohydrolase II are fused
to ribBA in V. fischeri. To answer these questions, we
examined whether in this species the rib operon is present,
and if so, whether ribBA is present in the fused form.
We tried to amplify a partial ribD sequence by PCR using

andevenifribB does not work, ribBA seems to complement
this defect.
DISCUSSION
In this study, we reconfirmed that the light emitted from
P. phosphoreum IFO 13896 is largely diminished in 1%
NaCl medium as compared with that in medium containing
3% NaCl (Fig. 2, B1 and B2). Although the mutant M1
emitted light sparingly even in the logarithmic growth phase,
the strain grew quite well (Fig. 2, B3). In the past, it was
difficult to maintain strongly light emitting strains on a slant
because they gradually changed in storage. We speculate
that this change is caused by reverse mutations as follows.
The strains, which are available from depositories, are
usually strongly light emitting because they were screened
based on this criterion. However, such strains seem to be
unusual, while dim or dark mutants seem to be common.
Once a mutation occurs in a light emitting strain, dim or
dark mutants prevail rapidly as if they grow faster and more
densely than the parent. The evidence above supports that
the function of the lux operon is not to produce light, and
light is a by-product of the bacterial luciferase [17,30].
Van den Berg et al. divided species bearing hcp into three
classes [24]. According to their classification, P. phosphoreum
is in class II. They reported that the spacing of the N-terminal
cysteines in this class is CX
2
CX
11
CX
6

AE011704 and AE012168), two genes, ribA and ribB,are
fused as ribBA and the rib genes are organized in a complete
operon, although a gene may be inserted between ribD and
ribC in Xanthomonas group (Fig. 4).
The function of ribX is not yet clear. Although the gene is
not present in Buchnera, it appears just upstream of ribD in
Pasteurellaceae and is present at this location in all other
species in the Proteobacteria c-subdivision, although a gene
may be inserted between ribX and ribD in the Xanthomonas
group. However, the genes found just upstream of ribX in
these species are quite variable and not arranged in one
direction, as shown in Fig. 4. This may indicate that ribX is
relatedtotherib operon. The gene is not very long and its
product is rich in basic amino acids. P. phosphoreum RibX
was calculated to have a pI of 7.6 suggesting that the protein
could be a DNA binding protein. In B. subtilis,regulation
of the biosynthesis of riboflavin has been studied intensively.
FMN has been identified as the effecter molecule for
regulation of the rib operon [1,32,33] and the cis-acting
region has been identified as ribO [1]. However, just
upstream of ribD in B. subtilis, no regulatory gene is present
and the trans-acting protein(s) has not been identified in any
other location [1]. To date, genome sequences of 19 species
of Gram-positive bacteria have been deposited in the
database; 12 of these species are riboflavin autotrophic
and seven are auxotrophic. In autotrophs, rib genes
are organized in an operon, although in three species
of Mycobacterium (MTCY21B4, AE007016 and
MLEPRTN2), some genes are inserted in the rib operon.
In all of these species, ribX is present not just upstream of

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