Báo cáo Y học: 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 pot

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 region, using SUGDAT, Sequencing Using Genomic DNA As a Template. We could not find this gene but found a gene for hybrid-cluster protein (prismane protein). To find ribD in a different region, a partial ribD sequence was amplified and sequenced using a PCR-based method, and subsequently the genomic DNA was sequenced in both directions from this partial sequence using SUGDAT. Because ribC was found just downstream of ribD, we sequenced further downstream of ribC and confirmed that another complete set of rib genes, ribD, ribC, ribBA,andribE, is present in P. phosphoreum. The presence of a complete rib operon in P. phosphoreum explains why this species can synthesize flavins at enhanced levels to sustain a strong light emission. Furthermore, we sequenced the rib operon in Vibrio fischeri, another repre- sentative luminous bacterium, in a manner similar to that described above, and confirmed that a complete operon is present also in this species. The organization of rib genes in an operon in the Proteobacteria c-subdivision is discussed. Keywords: biosynthesis of riboflavin; hybrid-cluster protein; Photobacterium phosphoreum; rib operon; SUGDAT. In bacteria and archaea, a complete set of genes for riboflavin biosynthesis, rib genes, was first identified in Bacillus subtilis [1]. Four enzymes participate in the synthesis of riboflavin from GTP and ribulose 5-phos- phate: pyrimidine deaminase/pyrimidine reductase, riboflavin synthase, GTP cyclohydrolase II/3,4-dihydr- oxy-2-butanone 4-phosphate synthase, and lumazine syn- thase. The genes coding for these enzymes are designated ribG, ribB, ribA and ribH, respectively, in B. subtilis. But in Escherichia coli, the genes for GTP cyclohydrolase II 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. Fax: +81 6 6605 2769, Tel.: +81 6 6605 2783, E-mail: kasai@bioa.eng.osaka-cu.ac.jp Note: The nucleotide sequences reported in this paper have been submitted to DDBJ/EMBL/GenBank nucleotide sequence database and are available under the accession numbers AB065117, AB065118 and AB076605. (Received 16 July 2002, revised 8 September 2002, accepted 10 October 2002) Eur. J. Biochem. 269, 5851–5860 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03304.x Photobacterium phosphoreum IFO 13896 is a strongly light emitting strain. The strong emission of light in this bacterium is primarily attained by the activated expression of the lux (luminescence) operon, by which luciferase and tetradecanal, one of the substrates of luciferase, are supplied in large quantities. Furthermore, the stimulated biosynthesis of flavins seems to be necessary for the strong emission because FMN is another substrate of luciferase. The colour of the cells is a deeper yellow when the strain is strongly emitting light and so this inference appears correct. Because luminous bacteria are classified as Gram-negative, this stimulated synthesis of riboflavin seems to contradict the concept described above. Lee et al. reported that in P. phosphoreum,alloftherib genes except ribD are clustered just downstream of the lux operon [7]. However, this cluster is not a complete rib operon, the presence of which seems to be absolutely necessary for the efficient regulation of riboflavin biosynthesis at the transcriptional level in this strain. Recently, the genome sequences of Vibrio cholerae [8] and Pseudomonas aeruginosa [9] were released. The rib genes are organized in an operon in both cases although the two 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 did not exceed 10. One litre of the medium contained: 2 g polypeptone (Nihon Pharmaceutical); 1 g yeast extract (Oriental yeast); 6 g glycerol; 1 g KH 2 PO 4 ; 660 mg NaOH; and 30 g or 10 g NaCl for 3% or 1% NaCl medium, respectively; pH of the medium was 7.0. The parent strain and three mutants were precultured in the respective media until a turbidity of 2–6 was attained. The seed cultures (150–500 lL) were transferred to 10 mL respective medium in L-shaped culture tubes 15 mm in diameter and 165 mm long, and incubated at 20 °C with shaking. Growth was monitored by measuring turbidity at 650 nm with a 1-mm light path cuvette, inserting a red glass filter (Toshiba, R-63) between the cuvette and detector to cut off the light emitted by bacteria. Bioluminescence was measured in arbitrary units at a distance of 5 mm from the culture tube in a dark box using a photodiode (Hamamatsu Photonics, S2281) connected to an amplifier (Hamamatsu Photonics, C2719). A curve of the bioluminescence plotted as a function of time was designated the bioluminescence curve. Measurement of the total amount of flavins To obtain faster growth, the concentrations of polypeptone and yeast extract in the media used for this experiment were increasedto5and2.5gÆL )1 , respectively. To determine the total amount of flavins in the respective exponential cells, 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]. A solution of FP 390 was heated at 95 °Cfor5minto denaturate protein and the precipitate was removed by centrifugation at 16 000 g for 5 min. The concentration of P-flavin in the supernatant was 18.4 pmolÆmL )1 assuming that the extinction coefficient at 448 nm of this flavin is equal to that of riboflavin (12.3 m M )1 Æcm )1 [16]). Although this concentration was about four times the maximum measurable by fluorometry, the chloroform extract of the photolysed sample showed only negligible fluorescence. So, we concluded that P-flavin cannot be quantified using the lumiflavin fluorescence method and P-flavin is not included in the flavins quantified by this method. DNA sequencing The genomic DNA of P. phosphoreum IFO 13896 or V. fischeri ATCC 7744 was prepared using Genomic-tip 500/G (QIAGEN) as previously reported [17]. PCR was 5852 S. Kasai and T. Sumimoto (Eur. J. Biochem. 269) Ó FEBS 2002 conducted using the ASTEC program temperature control system PC-701 with 0.2 mL PCR tubes. The genomic DNA was used as a template. The oligonucleotides used for primers were purchased from Genset. On preparation of the 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. A 1995-bp sequence downstream of the rib gene cluster reported by Lee et al. [7] was determined by performing SUGDAT seven times as outlined in Fig. 1A. The first primer, 5¢-CGAAAGCTTCTCATGGCCGC-3¢,was designed by referring to the reported sequence (DDBJ/ EMBL/GenBank database accession number, L11391). Sequencing of the complete rib operon with both flanking regions in P. phosphoreum IFO 13896 (5616 bp) is outlined in Fig. 1B. Two conserved amino acid sequences in RibD, GATAYVT and (L/I)WVEAGS, were found by comparing RibD sequences from V. cholerae (AE004298), E. coli (X64395) and H. influenzae (U32775), and according to these sequences, two degenerate primers, 5¢-GGN GCIACNGCITAYGTNAC-3¢ (where I ¼ inosine) and 5¢-NGVICCNGCYTCIACCCANA-3¢, were designed. Using these primers, a 720-bp DNA sequence was amplified and the nucleotide sequence was determined. To determine a 1379-bp sequence upstream of the partial ribD,the genomic DNA was sequenced by five rounds of SUGDAT. A 641-bp sequence downstream of the partial ribD was determined by two rounds of SUGDAT and it was found that ribC overlaps with the tail of ribD. To confirm the presence of ribBA downstream of ribC, a conserved amino acid sequence in RibBA, NDDGTMA, was found by comparing RibBA sequences from V. cholerae (AE004298), P. phosphoreum (RibBA¢) (L11391), and P. leiognathi (M90094), and a degenerate reverse primer, 5¢-GCCATNG TICCRTCNTCRTT-3¢ was designed according to this amino acid sequence. Using this primer and a forward one, we amplified an 853-bp sequence and sequenced this 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 arrows indicate sequences determined in these orientations by SUG- DAT. Solid bars indicate PCR products. (A) The rib gene cluster was found downstream of the lux operon in P. phosphoreum by Lee et al. [7]. Note that ribD is not present in this cluster. A region downstream of the lux–rib cluster in P. phosphoreum was sequenced by SUGDAT seven times and a 1995-bp sequence was determined in which a gene for the hybrid-cluster protein (prismane protein), hcp, was found. The scaleisonlyeffectivefortherib gene cluster and hcp. (B) A partial ribD sequence was amplified by PCR using two degenerate primers. A 1379- bp upstream sequence was determined by five rounds of SUGDAT and a 641-bp downstream sequence was determined by two rounds. Because at this sequencing stage, a new rib operon seemed to be present in this region, two DNA sequences were amplified using two pairs of primers, degenerate and nondegenerate. An 807-bp further down- stream sequence was determined by three rounds of SUGDAT. (C) A 5232-bp sequence of a rib operon with both flanking regions in V. fischeri was determined using basically the same strategy as des- cribed above. Ó FEBS 2002 Stimulated synthesis of flavins in P. phosphoreum (Eur. J. Biochem. 269) 5853 P. phosphoreum, and the corresponding region (5232 bp) of V. fischeri, determined as above, will appear in the DDBJ/ EMBL/GenBank nucleotide sequence database with the accession numbers, AB065117, AB065118 and AB076605, respectively. RESULTS Stimulated biosynthesis of flavins in P. phosphoreum IFO 13896 strongly emitting light P. phosphoreum emits light strongly when cultured in a medium containing 3% NaCl but only weakly in 1% NaCl [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. The parent strain emitted  37 times more light in medium containing 3% NaCl than it did in medium containing 1% NaCl. This drastic diminution of bioluminescence caused by a decrease of ionic strength in the medium has been reported previously [18–20]. M1 is a dark mutant and does not emit light detectable with the naked eye. M2 and M3 showed about 1/60 and 1/10 the specific bioluminescence of the parent strain cultured in 3% NaCl, respectively. To confirm that strongly light emitting cells produce large amounts of flavins, we determined the total amount of flavins in cells from the parent strain cultured in 3% or 1% NaCl, and from three mutants cultured in 3% NaCl. Respective values are shown in Table 1. We determined the Fig. 2. Growth and bioluminescence curves of the parent and three mutant strains of P. phosphoreum IFO 13896. Five growth curves are collectively showninpanel(A)forcomparison.j,Parentstrainculturedin3%NaClmedium;d, parent strain in 1% NaCl; m, mutant strain M1 cultured in 3% NaCl; .,M2in3%NaCl;r,M3in3%NaCl.Growth(h) and bioluminescence (s) curves of the parent strain in 3% and 1% NaCl are shown in panel B1 and B2, respectively, and those of M1, M2 and M3 in 3% NaCl are shown in B3, B4 and B5, respectively. 5854 S. Kasai and T. Sumimoto (Eur. J. Biochem. 269) Ó FEBS 2002 total amount of flavins using the lumiflavin fluorescence method. The term ÔflavinsÕ refers to riboflavin, FMN and FAD. P-flavin, which is contained in the extract of luminous bacterial cells [12], is not included because it is not photolysed to a fluorescent lumiflavin-type product, as described above. Because M1 emits light sparingly, this mutant seems to produce flavins at a basal level. The parent strain cultured in 3% NaCl, meanwhile, produced about eight times more flavin than M1 cells. Considering that the parent cells produce P-flavin, it is appropriate that they produce more flavin. The parent cells in 1% NaCl, and the M2 and M3 cells in 3% NaCl, produced about 2, 1.5- and 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 C-terminal region of the E. coli hybrid-cluster protein, Hcp (prismane protein). To confirm unequivocally the presence of the gene for this protein, we performed SUGDAT a further six times. In the determined sequence (AB065117), an open reading frame of 1662 bp was located 80 bp downstream of the termination codon of ribA¢ in the opposite direction. Hcp was originally found in Desulfovibrio vulgaris and named prismane protein because it was postulated that the protein contains the [6Fe)6S] cluster [22]. Three-dimen- sional structural analysis revealed that the protein does not contain this cluster but has two Fe/S clusters, one of which is a hybrid [4Fe)2S)2O], and was consequently renamed hybrid-cluster protein [23]. Recently, E. coli hcp was over- expressed in a recombinant E. coli and it was shown that the protein has properties similar to those of D. vulgaris Hcp [24]. A homology search using FASTA revealed that it is present in 22 species as of today. In addition, K € uuhn et al. reported that a putative protein displaying 55% homology to the D. desulfuricans Hcp is present in Morganella morganii [25]. The number of amino acid residues compo- sing each of the E. coli [24], Salmonella typhimurium (AE008739), Salmonella enterica (AL627268), Yersinia pestis (AJ414147), Acidithiobacillus ferrooxidans (TFU73041), D. vulgaris [22], and D. desulfuricans [26] Hcp is 545–556 and comparable with that of the P. phosphoreum Hcp (553 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 assumed that the remaining gene, ribE, would be present downstream of ribBA and confirmed this in the same way as above (Fig. 1B). Further, we determined 1379-bp upstream and 855-bp downstream sequences by SUGDAT as described above. In the sequence shown in Fig. 3, six complete and two partial open reading frames were found. The amino acid sequences deduced from the nucleotide sequences of these open reading frames are also shown in Fig. 3. At the 5¢-end, a partial glyA sequence, which encodes serine hydroxy- methyl transferase, was found in the same orientation as the rib operon. A gene of 450 bp was found 217 bp downstream of the termination codon of glyA. This gene is conserved and generally found just upstream of ribD in Gram-negative Table 1. Specific bioluminescence emitted and total amount of flavins produced. P. phosphoreum strain Parent Parent M1 M2 M3 Concentration of NaCl in the medium 3% 1% 3% 3% 3% Specific bioluminescence (arbitrary units/turbidity) 18 401 515 2 315 1840 Total amount of flavins (pmol/mL turbidity) 348 86 45 72 120 Ó FEBS 2002 Stimulated synthesis of flavins in P. phosphoreum (Eur. J. Biochem. 269) 5855 5856 S. Kasai and T. Sumimoto (Eur. J. Biochem. 269) Ó FEBS 2002 bacteria, whether or not the rib genes are organized in an operon. Because it seems to be closely related to the rib operon, we designated it ribX and will discuss its function later. Downstream of ribX, four rib genes ) ribD, ribC, ribBA and ribE ) were organized in a complete operon in thesamearrangementasthecompleterib operons in other species. The tail of ribD overlapped with the head of ribC (Fig. 3) and the number of amino acid residues of RibD in this species is larger by about 20 residues than the numbers 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. reported that both genes are present together in He. pylori, but ribBA did not complement the ribA mutation in E. coli [28]. Downstream of the rib operon, a complete nusB,which encodes an antitermination factor, and a partial thiL,which encodes thiamine phosphate kinase, were found. The array of three genes, ribE, nusB and thiL, is conserved widely in bacteria classified in the Proteobacteria c-subdivision (Fig. 4). Identification of a complete rib operon in V. fischeri ATCC 7744 and analysis of the determined 5232 bp sequence Callahan and Dunlap reported that in V. fischeri,a gene encoding 3,4-dihydroxy-2-butanone 4-phosphate Fig. 4. Comparison of the arrangement of the rib genes in bacteria classified in the Proteobacteria c-subdivision and Gram-positive bacteria. In Enterobacteriaceae ribX, ribD and ribE are organized in a cluster but ribC and ribBA are not. The latter is not fused but remains as two separate genes, ribA and ribB. In Vibrionaceae, Pseudomonadaceae and the Xanthomonas group, the rib genes are organized in a complete operon and two genes, ribA and ribB, are fused as one, ribBA. In Gram-positive bacteria, four rib genesareorganizedinanoperonbut ribX is separate. Neighbouring genes are bound with solid lines and separated genes with dashed lines. Arrows under the genes indicate the orientation in which the respective genes are arranged. The orientation of separated genes is not indicated because it changes depending on the species. Table 2. Homologies of five rib proteins, RibX, RibD, RibC, RibBA and RibE of P. phosphoreum or V. fischeri with those of seven species. Amino acid 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 the two degenerate primers used to amplify the gene of P. phosphoreum. The DNA sequence was amplified success- fully and a 5232-bp sequence of a complete rib operon with both flanking regions was determined as shown in Fig. 1C. In the sequence determined, six complete and two partial open reading frames were found. At the 5¢-end of the sequence, a partial 430 bp gene of unknown function (ORF1) was found in the opposite orientation to the rib operon. Although we performed a homology search on this partial protein using FASTA , no homologues were found. Downstream of this open reading frame, seven genes, ribX, ribD, ribC, ribBA, ribE, nusB and thiL, were arranged in the same sequence as that in P. phosphoreum. Homologies of the five rib gene products of V. fischeri with those of five different species, along with RibC¢,RibBA¢ and RibE¢ of P. phosphoreum and P. leiognathi are shown in Table 2. Based on these results, we concluded that a complete rib operon, in which ribBA is fused as one gene, is present also in V. fischeri. Callahan and Dunlap constructed a mutant of V. fischeri MJR1 defective in ribB, and examined it for altered growth and bioluminescence in comparison with the parent strain [29]. However, the mutant showed no difference, regardless of the presence or absence of exogenously supplied ribofla- vin. They concluded that RibB apparently is not required for, and does not play a significant role in normal light production. Their conclusion is appropriate because also in V. fischeri,acompleterib operon is present as shown above 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 C and this spacing was found in the amino acid sequence of P. phosphoreum Hcp. Although the bacteria classified in class II are also classified in the Proteobacteria c-subdivision, hcp is present nonubiqui- tously in bacteria classified in this subdivision and was not detected even in the genome of V. cholerae. Evidence that hcp is present in P. phosphoreum in addition to E. coli, S. typhimurium, S. enterica, Y. pestis and M. morganii, among bacteria in this subdivision, strongly supports that the luminous bacteria should be placed with enteric bacteria of sea animals. It was suggested that Hcp has a role in nitrate/ or nitrite respiration [24], and overproduction of the protein is toxic to the cells of Clostridium perfringens in the presence of oxygen [31]. Comparing the arrangement of rib genes in bacteria classified in the Proteobacteria c-subdivision, we can deduce the process of organization of these genes. Among these species, in Buchnera, rib genes are scattered and not organized at all (NC 002528). In two species of Pasteurell- aceae, H. influenzae and Pasteurella multocida, rib genes are not yet organized but there are indications of organization. The ribX gene is present upstream of ribD in these species (U32775 and AE006112). In four species in Enterobacteri- aceae, E. coli (X64395), S. typhimurium (AE008714), S. ent- erica (AL627266) and Y. pestis (AJ414155), ribX, ribD and ribE are organized in a cluster but ribC and ribBA are not as shown in Fig. 4. The latter is not fused but remains as two divided genes, ribA and ribB. Finally, in Vibrionaceae (AE004298), Pseudomonadaceae (AE004821 and AE004822) and the Xanthomonas group (AE003934, 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 the rib operon but far from the operon. For example, in B. subtilis, ribX is designated as ytcG (Z99118). In four 5858 S. Kasai and T. Sumimoto (Eur. J. Biochem. 269) Ó FEBS 2002 auxotrophs, ribX is not present but it is in three, Listeria innocua (AL596169), Listeria monocytogenes (AL591979) and Streptococcus pyogenes (AE006498). On the basis described above, we suggest that RibX may be a regulator for the rib operon. In Gram-positive bacteria, ribX may move to another position because it may acquire additional function(s). For the same reason, the gene may be present even in auxotrophs. We have no experimental evidence of this but we consider that it is worth examining whether the gene works as expected, because in none of the species have regulatory gene(s) for the rib operon been identified in spite of a great deal of effort. ACKNOWLEDGEMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are indebted to T. Nakamura of the Graduate School of Science, Osaka City University, for assistance in operating the DNA sequencer. REFERENCES 1. Perkins, J.B. & Pero, J.G. (1993) Biosynthesis of riboflavin, biotin, folic acid, and cobalamin. 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Coquard, D., Huecas, M., Ott, M., van Dijl, J.M., van Loon, A.P. & Hohmann, H.P. (1997) Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction. Mol. Gen. Genet. 254, 81–84. 33. Mack, M., van Loon, A.P. & Hohmann, H.P. (1998) Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC.J.Bacteriol.180, 950–955. 5860 S. Kasai and T. Sumimoto (Eur. J. Biochem. 269) Ó FEBS 2002 . 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. discussed. Keywords: biosynthesis of riboflavin; hybrid-cluster protein; Photobacterium phosphoreum; rib operon; SUGDAT. In bacteria and archaea, a complete set of genes for riboflavin biosynthesis, rib. ) ribD, ribC, ribBA and ribE ) were organized in a complete operon in thesamearrangementasthecompleterib operons in other species. The tail of ribD overlapped with the head of ribC (Fig. 3) and