Photobacterium mandapamensis: Its got the lux F gene

Bioluminescence is the production and emission of light by a living organism. In this Paper Kaeding et al (2007) used a multigene phylogenetic approach and examined a wide diversity of deep-water and shallow-water dwelling bioluminescent animals for the presence of Photobacterium mandapamensis as a light organ symbiont. Photobacterium mandapamensis is found in the light organs of many fish and squid. There are several species of bioluminescence bacteria that have been documented as symbionts;  Photobacterium kishitanii, Photobacterium leiognathi, Photobacterium mandapamensis, Vibro fischeri and Vibro logei.  Photobacterium leiognathi is a light organ symbiont of Leignathid fish and shares various taxonomic traits with Photobacterium mandapamensis, these are separated by the luminescence genes luxAB(F)E.

Five species of bioluminescent hosts were collected and their light organs were examined. The host specimens were collected from shallow-deep continental shelf locations in the Pacific, Atlantic and Indian oceans. They were collected from a variety of fish markets on the day of capture, the locations are listed in table 1.

The mitochondrial DNA was extracted from the fish specimen’s flank muscle tissue. The flank muscle tissue was removed from skin and stored in 90% ethanol at 20˚C. QIAGEN QIAquick tissue extraction was used to extract DNA from small chunks of the tissue. The specimen’s light organs were dissected. The fish were kept chilled on ice until dissection, within an hour or two of collection. The ventral light organ present in Acropoma japonnicum, Siphamia vesicolor and Gadella jordani or the circumesophageal light organ in Photopectoralis panayensis and Photopectoralis bindus were aseptically dissected from the fish and homogenized in 0.5ml or 1.0ml of sterile artificial 70% seawater containing 25 mM HEPES buffer (BSW-70) in a sterile handheld Tenbroeck glass tissue grinder.

The light organ homogenates, which are suspended cell fragments were then serially diluted in BSW-70 and portions of one or more of the end dilutions were spread on plates of LSW-70 agar, Plates were incubated for 18 to 24h at room temperature. Nonluminous colonies were not observed. 10-40 colonies were then picked at random form plates for each specimen, purified on LSW-70 agar plates and stored as viable culture at 75˚C in cryoprotective (this prevents freezing damage to the tissue) medium in the laboratory’s permanent strain collection.

Genomic DNA was purified from 1-ml cultures of strains grown overnight in LSW-70 broth by using the QIAGEN DNeasy tissue extraction kit and by following the manufacturers protocol for gram-negative bacteria.

The gyrB, luxAB(F)E and ribBHA genes were amplified by PCR using Tag polymerase along with reagents from either the Eppendorf master tag kit or the promega tag DNA polymerase kit. DNA genomic profile analysis was carried out with repPCR on purified genome DNA of individual strains using primers REP1R-I and REP2-I.

The template for the reactions was prepared directly from cell pellets resuspended in 50µl of sterile deonised water and repelleted to remove the supernatant, this was then amplified. Provisional species identifications were based on 90% or greater identity to sequence of the luxA amplicon or the luxA-luxB of Photobacterium mandapamensis or Photobacterium leiognathi from GenBank.

The sequencing and phylogenetic analyses involved the staff of the University of Michigan sequencing core using the respective amplification primers and dye terminator cycle to sequence the PCR products. This was carried out on a Perkin-Elmer ABI3730 DNA analyser.  The bacterial phylogeny was reconstructed by parsimony analysis preformed with PAUP (phylogenetic analysis using parsimony). For non-luminous taxa, luxABFE sequences were treated as missing data.

To construct the fish phylogeny, mitochondrial genes encoding 16S rRNA and cytochrome oxidase subunit were amplified and sequenced using the primer sequences and PCR protocols. Sequence data were analysed by direct optimization as implemented in OY (POY without parallel options). POY is a program for cladistic and phylogenetic analysis using sequence and/or morphological data.

All five species of fish were analysed for the presence of light organ symbionts, particularly identifying what specific strains of bacteria were present. The results revealed three phylogenetically distinct clades; Photobacterium mandapamensis clade I and clade II and Photobacterium leiognathi.

Acropoma japonnicum

Previous studies have identified Photobacterium leiognathi as the exclusive symbiont of Acropoma japonnicum however the presence of the lux F gene confirms that, the AJ-1a strain found in this species was a strain of Photobacterium mandapamensis. Acropoma japonnicum had substantial bacterial diversity, with at least three strain types present in the specimen Ajapo.2. The gyrB and luxAB(F)E genes of strains representative of each type were sequenced. The strains ajapo 2.1, ajapo 2.6 and ajapo.2.19 were identified as Photobacterium mandapamensis clade I and clade II and Photobacterium leiognathi.

Photopectoralis panayensis and Photopectoralis bindus

The results revealed a cosymbiosis of Photobacterium mandapamensis and Photobacterium leiognathi in these two leiognathid fishes. The identification of Photobacterium mandapamensis in the 9/43 strains examined demonstrates that Photobacterium leiognathi is not the exclusive symbiont of leiognathid fishes.

Siphamia vesicolor and Gadella jordani

The light organ symbionts showed phylogenetic clustering; some strains of Photobacterium mandapamensis identified in both species were all members of clade II. Six strains found in Siphamia vesicolor and eight strains found in Gadella jordani. In these two species it appeared that Photobacterium mandapamensis was the sole symbiont present.

The authors interpreted the data believing that there was evidence of cospeciation. They tested a cospeciation hypothesis using mitochondrial 16S rDNA and COI gene sequences to reconstruct phylogeny of the fishes with the symbiont Photobacterium mandapamensis and compared it to the phylogeny of representative bacterial strains from these fishes. The clade structure did not topologically match. Therefore the hypothesis is refuted and other factors must determine the host preference in Photobacterium mandapamensis. The phylogenetic diversity showed that Photobacterium mandapamensis is a general light organ symbiont because of its wide host range. The difference in host ranges of Photobacterium mandapamensis and Photobacterium leiognathi indicate that these bacteria might differ in other ways as well. Differences like environmental distribution and their physiology would therefore relate to the hosts they prefer to colonize. No phylogenetically intermediate strains have been found. This indicates that despite the traits Photobacterium mandapamensis and Photobacterium leiognathi share they are biologically distinct, and perhaps prone to competition, the predominance of Photobacterium mandapamensis in Acropoma japonicum and of Photobacterium leiognathi in Photopectoralis panayensis and Photopectoralis bindus suggests that competition could change the presence or number of one bacterial species within the individual light organs.

It was also discovered that distantly related fishes Acropoma japonnicum and Gadella jordani harbour the same bacterial species. Closely related fishes Acropoma japonnicum and Alteromonas hanedai and Gadella jordani and Parribacus japonicus harboured different bacterial species. Some fish Acropoma japonnicum, Photopectoralis panayensis and Photopectoralis bindus harbour members of two/three different bacterial clades. The main factors that could determine these bacteria preferences are that the fish do not discriminate between the phenotype of the bacteria or that environmental distribution results in chance symbiosis of a particular species.

The authors mention that the bacterial symbionts of Gadella jordani and Photopectoralis panayensis previously had not been examined and Photopectoralis bindus only a single bacterial strain from the light organ of a single specimen had been identified. This could means that more sampling will be needed to confirm the results from this study.

This paper interested me initially because it looked into the presence of bioluminescence in light organs of fish and squid; however I became more intrigued by the author’s interpretation of the data and whether there was a possibility for co-evolution between bacteria and fish species. This data might have contradicted their hypothesis but it could be possible that other species may have co-evolved at one point in their evolutionary history. This paper also shows the impacts of mistakes in identification for instance previous studies have identified Photobacterium mandapamensis as Photobacterium leiognathi and this implied that only Photobacterium leiognathi was the light organ symbionts of leignathid fishes, when in fact this study has evidenced the presence and cosymbiosis of Photobacterium mandapamensis in Leiognathid fish.

Kaeding A.J, Ast  J.C, PearceM.M, rbanczyk H, Kimura S, Endo H, Nakamura M, Dunlap P.V (2007) Phylogenetic Diversity and Cosymbiosis in the Bioluminescent Symbioses of “Photobacterium mandapamensis”, Applied and Environmental Microbiology, May 2007, p. 3173–318