TY - JOUR
AU - Honda, Takeshi
AB - Abstract The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens . Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae , the operons are located on chromosome 2. Photobacterium phosphoreum, Photorhabdus luminescens, transfer of the lux operon , Vibrio cholerae, Vibrio fischeri The number of species of luminous bacteria identified to date is only about 20 ( 1 ), and will not increase drastically because almost all species seem to have been identified as they can be detected readily by the bioluminescence they emit. All luminous bacterial species possess the lux operon, although the genes composing the operon vary depending on the species. The luminous species are widely distributed among γ-Proteobacteria considering their number: most luminous species are classified in Vibrionaceae , but several are also in Shewanellaceae , and even in Enterobacteriaceae ( 1 ). This feature of the distribution of the operon among bacteria seems to indicate that the operon was transferred independently of phylogeny, and means that it is difficult to find the pathway through which the operon was transferred, using a typical phylogenetic analysis. A comparative analysis of the two flanking regions of the operon in the respective species may be an alternative way to solve this problem because the operon usually seems to be transferred accompanying flanking genes. We can compare analytically genes flanking the lux operons of various species quite easily if the genomic sequences have been published. Genome sequencing in a variety of organisms has advanced dramatically in recent years. Now, genomic sequences of over 400 prokaryotic strains have been published and the number of completed genomic sequences increases every month. However, the genomic sequences of only two species of luminous bacteria, Photorhabdus ( Pr .) luminescens TT01 ( 2 ) and Vibrio fischeri ES 114 ( 3 ), have been published. Among the non-luminous species in Vibrionaceae , however, the genomic sequences of five species have been published: the sequence of V. cholerae N 16961 appeared in the DDBJ/EMBL/GenBank database in 2000 ( 4 ), that of V. vulnificus CMCP6 in late 2002 (accession numbers, NC 004459 and NC 004460), those of V. parahaemolyticus O3:K6 RIMD 2210633 ( 5 ) and V. vulnificus YJ016 ( 6 ) in 2003, and that of Photobacterium ( P .) profundum SS9 (NC 006370 and NC 006371) in 2005. The sequences of non-luminous species also provide valuable information, because we found the DNA sequence of the luminous strain V. cholerae NCIMB 41, to be quite similar to that of the non-luminous genome-sequenced strain V. cholerae N 16961 in overlapping regions ( 7 ). In this study, we started to sequence the regions flanking the lux operon in four species of luminous bacteria, V. cholerae MCIMB 41, Shewanella hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, aiming to find pathways of lateral transfer of the lux operon. MATERIALS AND METHODS Bacteria Six luminous bacterial species were used in this study. V. cholerae NCIMB 41 and S. hanedai NCIMB 2157 were obtained from NCIMB; V. fischeri ATCC 7744 from ATCC; P. phosphoreum IFO (NBRC) 13896 from the Graduate School of Engineering, Osaka City University; P. leiognathi NCIMB 2193 from Ocean Research Institute, The University of Tokyo; and V. harveyi B 392 from the Research Institute for Microbial Diseases, Osaka University. The respective strains were cultured at 23°C overnight with shaking in Luria–Bertani (LB) medium, containing 1% NaCl in the case of V. cholerae and 3% otherwise, and cells were harvested by centrifugation. PCR and DNA Sequencing Genomic DNA of the respective species for sequencing was prepared using Genomic-tip 500/G (QIAGEN) according to the supplier's manual. PCR and DNA sequencing were performed as described previously ( 8 , 9 ). A revised protocol for SUGDAT (Sequencing Using Genomic DNA As a Template) ( 7 ) was used. The lux operons as well as both flanking regions of four species were sequenced as follows, and the sequencing is summarized in Fig. 1 . Fig. 1. View largeDownload slide The lux operon and both flanking regions of four species, V. cholerae NCIMB 41, S. hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, along with the corresponding regions of the three genome-sequenced species, V. cholerae N 16961, Pr. luminescens TT01, and V. fischeri ES 114. Arrangement of genes in the lux operon and both flanking regions of four species, V. cholerae NCIMB 41, S. hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, and the sequencing of the respective species are summarized. Solid and open bars indicate PCR products amplified using non-degenerate and degenerate primers, respectively. Solid thin arrows indicate sequences determined in these directions by SUGDAT. Arrangements of genes of the corresponding regions of three genome-sequenced species, V. cholerae N 16961, Pr. luminescens TT01, and V. fischeri ES 114, are also shown over or below the one of V. cholerae NCIMB 41, and below the one of V. fischeri ATCC 7744, respectively, for comparison. Thick black arrows indicate the luxA to luxG genes, while open or gray arrows indicate other genes, in the respective directions. Solid lines connecting genes indicate non-coding sequences, while open ones in V. cholerae N 16961 and V. fischeri ES 114, show that those sequences are missing in respective species. In V. cholerae , comparing the corresponding regions of two strains, NCIMB 41 and N 16961, in the latter, twelve genes including the lux ones in the former are replaced with only two genes, VC1563 and VC1564, which are placed tentatively at the center of this region. Comparing two strains of V. fischeri , ATCC 7744 and ES 114, in the latter, a 3-kbp sequence is missing upstream of the lux operon and owing to this deletion, VfUp2 is omitted. Orthologues of five genes, VcUp1 to VcUp5 , upstream of the lux operon of V. cholerae NCIMB 41, were found as plu0793 to plu797, respectively, over 1.5 Mbp upstream of the lux operon in the opposite direction in the genome of Pr. luminescens TT01 and to show this arrangement, the respective genes were shaded and the compressed 1.5-Mbp sequence was hatched. Below the sequence of P. phosphoreum , a scale of 1000 bp is shown and this scale is applied for all sequences in this figure, except the compressed 1.5-Mbp sequence in Pr. luminescens . Fig. 1. View largeDownload slide The lux operon and both flanking regions of four species, V. cholerae NCIMB 41, S. hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, along with the corresponding regions of the three genome-sequenced species, V. cholerae N 16961, Pr. luminescens TT01, and V. fischeri ES 114. Arrangement of genes in the lux operon and both flanking regions of four species, V. cholerae NCIMB 41, S. hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, and the sequencing of the respective species are summarized. Solid and open bars indicate PCR products amplified using non-degenerate and degenerate primers, respectively. Solid thin arrows indicate sequences determined in these directions by SUGDAT. Arrangements of genes of the corresponding regions of three genome-sequenced species, V. cholerae N 16961, Pr. luminescens TT01, and V. fischeri ES 114, are also shown over or below the one of V. cholerae NCIMB 41, and below the one of V. fischeri ATCC 7744, respectively, for comparison. Thick black arrows indicate the luxA to luxG genes, while open or gray arrows indicate other genes, in the respective directions. Solid lines connecting genes indicate non-coding sequences, while open ones in V. cholerae N 16961 and V. fischeri ES 114, show that those sequences are missing in respective species. In V. cholerae , comparing the corresponding regions of two strains, NCIMB 41 and N 16961, in the latter, twelve genes including the lux ones in the former are replaced with only two genes, VC1563 and VC1564, which are placed tentatively at the center of this region. Comparing two strains of V. fischeri , ATCC 7744 and ES 114, in the latter, a 3-kbp sequence is missing upstream of the lux operon and owing to this deletion, VfUp2 is omitted. Orthologues of five genes, VcUp1 to VcUp5 , upstream of the lux operon of V. cholerae NCIMB 41, were found as plu0793 to plu797, respectively, over 1.5 Mbp upstream of the lux operon in the opposite direction in the genome of Pr. luminescens TT01 and to show this arrangement, the respective genes were shaded and the compressed 1.5-Mbp sequence was hatched. Below the sequence of P. phosphoreum , a scale of 1000 bp is shown and this scale is applied for all sequences in this figure, except the compressed 1.5-Mbp sequence in Pr. luminescens . In the case of V. cholerae NCIMB 41, we had already sequenced the lux operon with both flanking regions (AB115761), and then performed a further four rounds of SUGDAT downstream from the terminal of this sequence. The sequence in the fourth round showed 99% identity with that of VC1563 of strain N 16961 (AE004234) ( 4 ). Accordingly, to determine the further downstream sequence, we amplified a 2079-bp sequence by PCR, using non-degenerate primers, the reverse one designed referring to the sequence of VC1566 of strain N 16961 (AE004234), and sequenced the PCR product. The combined 3689-bp sequence downstream of the lux operon was registered as AB120061. On the other hand, because the lux operon seemed to be inserted between VC1562 and VC1563 in strain NCIMB 41, we tried to amplify a partial sequence of VC1562 using the genomic DNA of strain NCIMB 41 as a template by PCR, with non-degenerate primers, which were designed referring to the sequence of VC1562 of strain N 16961. This trial and then the next one to amplify a partial sequence of VC1561 were unsuccessful. However, we were finally successful in amplifying a partial sequence of VC1560 of strain NCIMB 41. Because we had already performed three rounds of SUGDAT upstream of the lux operon at this stage, we tried to amplify a sequence between the upstream edge and VC1560 using non-degenerate primers. We were successful in amplifying a 3,694-bp sequence and sequenced this PCR product. To sequence further upstream, we amplified a 2068-bp sequence using non-degenerate primers and sequenced the product. Finally, we determined a 7649-bp sequence (AB119994) upstream of the lux operon in this strain. In the case of S. hanedai NCIMB 2157, two partial sequences of luxC and luxA were amplified using two pairs of degenerate primers, which were used to amplify respective sequences of V. cholerae NCIMB 41 ( 7 ), and then sequenced. A sequence between these ones was amplified by PCR and then sequenced. Two PCR products, which were amplified using primers designed either according to the sequence determined as above or referring to the sequence AB058949, were sequenced. We then performed 10 rounds of SUGDAT in both directions from the respective terminals and finally determined a 13,760-bp sequence (AB261992). Comparing our sequence with AB058949, we found seven nucleotide-replacements in the lux operon and a large discrepancy downstream of luxG. In the case of V. fischeri ATCC 7744, starting from the tail of luxR , we performed 12 rounds of SUGDAT and determined a 5884-bp sequence (AB105805). Although our sequence contained the sequence reported as the cnf1 gene ( 10 ), in the sequence (AF023157), four nucleotide-insertions, a nucleotide-deletion, and two nucleotide-replacements were found compared with our sequence, and because of this discrepancy VfUp2 is rather longer than that previously reported ( cnf1 ). To sequence downstream of the lux operon, we performed eight rounds of SUGDAT starting from the tail of luxG and determined a 7898-bp sequence (AB107370). In the case of P. phosphoreum IFO 13896, because we have already determined the nucleotide sequence of luxF and both flanking regions ( 11 ), we performed 25 and 4 rounds of SUGDAT upstream and downstream from this region, respectively, and determined a 17,720-bp sequence in all (AB104437). Meanwhile, we performed 13 rounds of SUGDAT from the head of hcp toward the opposite direction, and determined a 7110-bp sequence (AB107988). Pulsed-field Gel Electrophoresis (PFGE) Five species of luminous bacteria were cultured as described earlier. Cells were harvested by centrifugation, embedded in 0.8% agarose gels, and processed prior to electrophoresis as described previously ( 12 , 13 ). PFGE was carried out on a CHEF Mapper system (Bio-Rad) with 0.8% agarose gel in 1.0× TAE buffer, pH 8.0. After electrophoresis, DNA bands were stained with ethidium bromide. Chromosomes of Hansenula wingei and Schizosaccharomyces pombe were obtained from Bio-Rad and used as size markers. Southern Hybridization Analysis After electrophoresis, DNA bands were transferred onto a nylon membrane. A 461-bp sequence in luxA was selected to prepare probes for detection of the lux operon. The following five pairs of primers were used to amplify the sequences: 5′-TGTTTCTCATATCAGCCTCC-3′ and 5′-GGGTAAACGTTTACATCTGG-3′ for P. phosphoreum ; 5′-TGTTTCTCATACCAACCACC-3′ and 5′-GGATAAACATTGACATCTGG-3′ for P. leiognathi ; 5′-TGTTTTTCGTATCAACCACC-3′ and 5′-GGATACACATCTACATTAGG-3′ for V. fischeri ; 5′-GGATTCAGTTGGATTTTCGG-3′ and 5′-GGATTCAGTTGGATTTTCGG-3′ for V. harveyi ; and 5′-TTACTAACCTACCAACCACC-3′ and 5′-GGTTGGACTTTCACTTCAGG-3′ for V. cholerae . The corresponding sequence was amplified by PCR to prepare a template for the preparation of a DIG-labelled probe in each species. The probe for the respective species was prepared with the above sequence using a PCR DIG labeling mix (Roche) according to the manufacturer's directions. DNA bands on the nylon membrane were hybridized with the probes and an image of the chemiluminescent bands was recorded on X-ray film. RESULTS AND DISCUSSION Sequence Analyses of both Flanking Regions of the lux Operons of Four Species V. cholerae NCIMB 41, S. hanedai NCIMB 2157, V. fischeri ATCC 7744, and P. phosphoreum IFO 13896, and Comparison of these Sequences with the Corresponding Ones of the Three Genome-sequenced species V. cholerae N 16961, Pr. luminescens TT01, and V. fischeri ES 114 In a previous study ( 7 ), we determined the nucleotide sequence of the lux operon of V. cholerae NCIMB 41. Because the genomic sequence of V. cholerae N 16961 had been published ( 4 ), we attempted to locate the operon in the genome. We sequenced both flanking regions of the operon as described earlier, and found that the operon is inserted between VC1560 and VC1563 ( Fig. 1 ). Five genes, VcUp1 to VcUp5 , are present upstream of the operon, and by homology search using BLAST ( 14 ) (blastp) at the National Center for Biotechnology Information, USA (NCBI), we found that the amino acid sequence deduced from the nucleotide sequence of VcUp1 shows 56% identity with that of plu0793 of Pr. luminescens TT01 ( 2 ), and further, the amino acid sequence deduced from VcUp2, VcUp3, VcUp4 and VcUp5 shows 60%, 47%, 38% and 49% identity with that from plu0794, plu0795, plu0796 and plu0797, respectively. Five genes, plu0793 to plu0797, are present also in tandem in the genome of Pr. luminescens , although they are over 1.5 Mbp upstream of the lux operon, as shown in Fig. 1 . By conducting BLAST analyses, we found that VcUp4 or plu0796 is an uncommon gene and no other orthologues are present in the database, but orthologues of the other four genes are distributed widely among bacteria and even archaea, although mainly among Gram-positive bacteria. The amino acid sequences deduced from these four genes show higher similarity scores with those of orthologues found in Gram-positive bacteria but less with those of Gram-negative ones. Meanwhile, five lux proteins of V. cholerae NCIMB 41 show high similarity scores with those of Pr. luminescens ( 7 ). Based on the earlier evidence, we deduced that the lux operon as well as the upstream cluster consisting of five genes was first transferred from a species of Gram-positive bacteria to V. cholerae . Thereafter, the five-gene cluster and the operon seem to have been transferred from V. cholerae to an ancestor of Pr. luminescens , although maybe indirectly, and further, either of the gene clusters may have been transferred to a new position 1.5 Mbp away from the original point in Pr. luminescens as shown in Fig. 2 . The reversed transfer of the gene clusters seems to occur rarely because two gene clusters can hardly be transferred from two different positions on a genome just to neighboring positions on a different genome. On the other hand, a cluster of the lux operon and its upstream genes might have been first transferred into an ancestor of Pr. luminescens from a species of Gram-positive bacteria and next transferred into Vibrio , while the cluster might have been split in a descendant of Pr. luminescens , however, this scheme of transfer seems to be improbable for the following reasons. If the cluster was first transferred to an ancestor of Pr. luminescens , the cluster should have spread widely in Enterobacteriaceae , into which Pr. luminescens is classified. In reality, the lux operon is distributed mainly in Vibrionaceae . Moreover, in Pr. luminescens only, the lux operon is partially broken because the luxG gene is deleted from the operon, as we will discuss below. Fig. 2. View largeDownload slide Transfer of the lux operon among luminous bacteria. The lux operons are shown with open black arrows, whereas the upstream five-gene clusters are shown with open gray arrows in V. cholerae, Pr. luminescence , and its ancestor. The sequence intervening between the operon and the five-gene cluster in Pr. luminescens is shown with a dotted line. Species, in which the lux operons are located on chromosome 2, are underlined. The lux operon and the upstream gene cluster seem to have been transferred from V. cholerae to an ancestor of Pr. luminescens , and further to Pr. luminescens , in which either of the gene clusters might have been transferred to a new position 1.5 Mbp away from the original point. Therefore, respective species were combined with an arrow in the direction from the former to the latter. Meanwhile, because the operon seems to have been transferred from either S. hanedai or V. fischeri to the other, the two species were combined with a line without a direction. Pathways of transfer of the operon among other luminous species could not be determined in this study. Fig. 2. View largeDownload slide Transfer of the lux operon among luminous bacteria. The lux operons are shown with open black arrows, whereas the upstream five-gene clusters are shown with open gray arrows in V. cholerae, Pr. luminescence , and its ancestor. The sequence intervening between the operon and the five-gene cluster in Pr. luminescens is shown with a dotted line. Species, in which the lux operons are located on chromosome 2, are underlined. The lux operon and the upstream gene cluster seem to have been transferred from V. cholerae to an ancestor of Pr. luminescens , and further to Pr. luminescens , in which either of the gene clusters might have been transferred to a new position 1.5 Mbp away from the original point. Therefore, respective species were combined with an arrow in the direction from the former to the latter. Meanwhile, because the operon seems to have been transferred from either S. hanedai or V. fischeri to the other, the two species were combined with a line without a direction. Pathways of transfer of the operon among other luminous species could not be determined in this study. For lateral gene transfer, two organisms must come into contact with each other. Because the habitats of strains of V. cholerae are unique among Vibrio species and terrestrial environments ( 7 ), two species of bacteria V. cholerae and Pr. luminescens , might have the chance to make contact with each other. We proposed that the function of the lux operon is to produce a halotolerant flavodoxin ( 8 , 15 ) and even in V. cholerae , the operon should have the same function ( 7 ). However, because Pr. luminescens is classified in Enterobacteriaceae and all species in this family inhabit terrestrial environments, it is improbable that the operon has the same function. We speculate that the operon acquired a new function when it was transferred to Photorhabdus and for this reason, the luxG gene might be lost from the operon. On the other hand, between the operon and VC1563, only one gene, VcDn1 , is present ( Fig. 1 ). Because the amino acid sequence deduced from the gene showed 47% identity with the sequences of two Shewanella species, ZP 00815380 and ZP 00905835, we expected an orthologue of this gene to be present downstream of the lux operon of S. hanedai , which had been reported to be luminous ( 1 ). We sequenced the operon with both flanking regions in S. hanedai NCIMB 2157 as previously described. We found three ORFs downstream of the operon but none of them was an orthologue of VcDn1 . Meanwhile, upstream of the operon, we also found three genes, two of which, are present just upstream of the operon, are orthologues of genes designated as luxI and luxR , which were also reported to be present just upstream of the lux operon in V. fischeri ( 16 , 17 ). Because we expected an orthologue of ShUp1 to be present downstream of luxR , we sequenced both flanking regions of the lux operon of V. fischeri ATCC 7744 as previously described. However, we could not find an orthologue of ShUp1 in the sequence determined and moreover, an orthologue is not present anywhere in the genome of V. fischeri ES 114. Except for V. fischeri, luxI and luxR were found in S. hanedai just upstream of the lux operon. This arrangement of genes indicates that the operon was transferred from V. fischeri to S. hanedai , and vice versa, although maybe indirectly. To show this relationship, we have connected the two species with a line without an arrow in Fig. 2 . In the case of V. cholerae , genes upstream of the lux operon show high similarity scores with orthologues present in Gram-positive bacteria as described earlier, whereas orthologues of the two genes, luxI and luxR , are widely distributed in Proteobacteria, regardless of bioluminescence, and consequently we speculate that the lux operon was first transferred by chance just downstream of luxI in a genome of a bacterium, very likely V. fischeri or S. hanedai , and further transferred to another bacterium, for example, V. fischeri or S. hanedai , along with luxI and luxR . We compared the sequence of the lux operon along with both flanking regions of V. fischeri ATCC 7744 with that of the corresponding region of strain ES 114. Although we determined a 7862-bp sequence downstream of luxR ( Fig. 1 ), the 1219 bp following luxR and the terminal 2725-bp sequence showed 87% and 95% identity with the corresponding regions of strain ES 114, respectively, and part of the intervening sequence of strain ATCC 7744 (3918 bp), showed only low identity with that of strain ES 114 (129 bp). In strain ES 114, owing to this reduction in the length of the sequence, VfUp2 , which is present in strain ATCC 7744, is deleted ( Fig. 1 ). In this strain, the deletion of genes seems to have occurred in many positions because the genome is about 1 Mbp smaller than that of strain ATCC 7744, as described subsequently ( Table 1 ). After the lateral transfer of gene clusters, such as the lux operon, the genes brought together seem to be deleted if they are unnecessary for the host: they seem to first be damaged partially and then be discarded stepwise. For this reason, upstream of the laterally acquired gene clusters, rather long non-coding regions seem to arise. In all cases in this study, such long non-coding regions were found upstream of the lux operon ( Fig. 1 ). On the other hand, the nucleotide sequence downstream of luxG of strain ATCC 7744, 5859-bp long, showed 92% identity with the corresponding sequence of strain ES 114. Table 1. Sizes of chromosomes 1 and 2 along with total sizes of five luminous bacteria as well as five genome-sequenced Vibrio strains and one Photobacterium strain. Lane  Species (Strains)  References  Sizes (Mbp)        Chromosome 1  Chromosome 2  Total  1  P. phosphoreum IFO 13896     3.2  1.8  5.0  2  P. leiognathi NCIMB 2193     3.3  1.6  4.9  3  V. fischeri ATCC 7744     3.2  1.9  5.1  4  V. harveyi B392     3.5  2.7  6.2  5  V. cholerae NCIMB 41     3.1  1.6  4.7    V. cholerae N 16961   ( 4 )   3.0  1.1  4.0    V. parahaemolyticus RIMD 2210633   ( 5 )   3.3  1.9  5.2    V. vulnificus CMCP6   AE016795 & AE016796  3.3  1.8  5.1    V. vulnificus YJ016   ( 6 )   3.4  1.9  5.3    V. fischeri ES 114   ( 3 )   2.9  1.3  4.2    P. profundum SS9   NC006370 & NC006371  4.1  2.2  6.3  Lane  Species (Strains)  References  Sizes (Mbp)        Chromosome 1  Chromosome 2  Total  1  P. phosphoreum IFO 13896     3.2  1.8  5.0  2  P. leiognathi NCIMB 2193     3.3  1.6  4.9  3  V. fischeri ATCC 7744     3.2  1.9  5.1  4  V. harveyi B392     3.5  2.7  6.2  5  V. cholerae NCIMB 41     3.1  1.6  4.7    V. cholerae N 16961   ( 4 )   3.0  1.1  4.0    V. parahaemolyticus RIMD 2210633   ( 5 )   3.3  1.9  5.2    V. vulnificus CMCP6   AE016795 & AE016796  3.3  1.8  5.1    V. vulnificus YJ016   ( 6 )   3.4  1.9  5.3    V. fischeri ES 114   ( 3 )   2.9  1.3  4.2    P. profundum SS9   NC006370 & NC006371  4.1  2.2  6.3  View Large Finally, we sequenced both regions flanking the lux operon in P. phosphoreum IFO 13896 as described earlier, to obtain information, to and from which species the operon was transferred. Upstream of the lux operon, five ORFs were found, and upstream of the hcp gene, three ORFs ( Fig. 1 ). However, none of them were orthologues of genes found in the flanking regions of the operon of the species mentioned earlier. By BLAST analysis, we found that most of them are located on chromosome 2 in the genome-sequenced five Vibrio and one Photobacterium strains, and the operon seems to be present on chromosome 2. Assignment of Loci of the lux Operons of Five Luminous Bacteria to either of Two Chromosomes Okada et al . ( 18 ) reported that vibrios commonly possess two chromosomes. In V. cholerae NCIMB 41, the lux operon is suggested to be located on chromosome 1 because as shown above, the operon is inserted between VC1560 and VC1563, which are located on chromosome 1 in strain N 16961, while in V. fischeri ES 114, the operon is located on chromosome 2 ( 3 ). In P. phosphoreum IFO 13896, the lux operon is deduced to be located on chromosome 2 as described earlier. We first confirmed that a two-replicon structure is common to the previous three species as well as two more typical luminous bacteria, P. leiognathi NCIMB 2193 and V. harveyi B 392. Intact genomic DNA of these five species was analysed using PFGE and two bands were found in all species examined, as shown in Fig. 3 . The sizes of the respective bands were estimated by comparing them with standard size markers. Sizes of chromosomes 1 and 2 along with total sizes of these five species as well as six genome-sequenced Vibrio and Photobacterium strains are summarized in Table 1 . Sizes of chromosome 1 of the respective species, except for P. profundum , are nearly the same. On the other hand, chromosome 2 varies in size over a wide range: that of V. cholerae N 16961 is 1.1 Mbp and much smaller than those of the others, whereas that of V. harveyi is 2.7 Mbp and far larger. Fig. 3. View largeDownload slide PFGE of intact genomic DNA of five species of luminous bacteria (left panel) and hybridization of the DNA with DIG-labeled DNA probes prepared based on partial luxA sequences (right panel). Lane 1, P. phosphoreum IFO 13896; lane 2, P. leiognathi NCIMB 2193; lane 3, V. fischeri ATCC 7744; lane 4, V. harveyi B 392; and lane 5, V. cholerae NCIMB 41. Genomic DNA of five species of luminous bacteria was subjected to PFGE and stained with ethidium bromide as shown in the left panel. The respective DNA bands were transferred to a nylon membrane and hybridized with DIG-labeled probes, and the hybridized bands were detected by the chemiluminescence emitted on decomposition of CSPD as shown in the right panel. Although only one of two bands in the respective lane was hybridized with the probe, in lane 1–4 in the right panel, in lane 5, a long smear hybridized band was observed. However, note that the band of chromosome 1 in lane 5 is also hybridized. Fig. 3. View largeDownload slide PFGE of intact genomic DNA of five species of luminous bacteria (left panel) and hybridization of the DNA with DIG-labeled DNA probes prepared based on partial luxA sequences (right panel). Lane 1, P. phosphoreum IFO 13896; lane 2, P. leiognathi NCIMB 2193; lane 3, V. fischeri ATCC 7744; lane 4, V. harveyi B 392; and lane 5, V. cholerae NCIMB 41. Genomic DNA of five species of luminous bacteria was subjected to PFGE and stained with ethidium bromide as shown in the left panel. The respective DNA bands were transferred to a nylon membrane and hybridized with DIG-labeled probes, and the hybridized bands were detected by the chemiluminescence emitted on decomposition of CSPD as shown in the right panel. Although only one of two bands in the respective lane was hybridized with the probe, in lane 1–4 in the right panel, in lane 5, a long smear hybridized band was observed. However, note that the band of chromosome 1 in lane 5 is also hybridized. Because the chromosomes of all five species examined were composed of two replicons, we next determined on which chromosome the lux operon is located in the respective species by hybridization with probes of DIG-labeled partial DNA sequences of luxA s. As shown in Fig. 3 , the operons of P. phosphoreum and V. fischeri were located on chromosome 2, as expected, and those of P. leiognathi and V. harveyi were also on the same chromosome. However, only the lux operon of V. cholerae NCIMB 41 was located on chromosome 1 consistent with the results obtained earlier. As shown earlier, chromosome 1 of the respective species is similar in size, and Heidelberg et al . ( 4 ) suggested that chromosome 1 of V. cholerae contains a larger fraction of genes known to be essential for growth. On the other hand, chromosome 2 varies in size drastically depending on the species and contains a larger fraction of hypothetical genes or genes of unknown function, which are less similar to the genes found in γ-Proteobacteria ( 4 ). The evidence that the lux operon is present on chromosome 2 in major species of luminous Vibrio and Photobacterium is consistent with that the operon is an uncommon gene cluster even in Vibrionaceae . In the previous section, we proposed that the lux operon and the upstream five-gene cluster had been transferred from Gram-positive bacteria to V. cholerae . Although in V. cholerae , the operon is located on chromosome 1, it might have been transferred from this species to chromosome 2 of other luminous vibrios. 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TI - Lateral Transfer of the lux Gene Cluster
JO - The Journal of Biochemistry
DO - 10.1093/jb/mvm023
DA - 2006-12-14
UR - https://www.deepdyve.com/lp/oxford-university-press/lateral-transfer-of-the-lux-gene-cluster-xYhzrJpTLN
SP - 231
EP - 237
VL - 141
IS - 2
DP - DeepDyve
ER -