Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan

Abstract

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.

FIG. 1.

Gene organization of the lux-rib₁ and lux-rib₂ operons of P. leiognathi. Genes and spacer regions are drawn to scale. Arrows above genes indicate the direction of transcription. Dashed rectangles between lumQ and luxC in P. leiognathi lux-rib₁ sequences indicate an approximately 200-bp region that is alignable to the lumP sequence of P. mandapamensis. Genes shaded gray have homologs in P. leiognathi, P. mandapamensis, and P. angustum. Small shaded rectangles outside of genes indicate noncoding intergenic sequences of P. leiognathi and/or P. mandapamensis that are alignable to sequences in P. angustum strains SKA34 and S14, including approximately 80 bp before the P. leiognathi putA start codon that aligns to a sequence within putA in P. angustum. Genes shaded dark gray in P. angustum indicate regions (orf5, orf6, and 759 bp from the start codon of putA) that are not alignable with a gene or intergenic sequence in P. leiognathi or P. mandapamensis. Double hash marks in the P. angustum sequences indicate contiguous sequences that have been separated to indicate the position of the lux-rib operon in P. leiognathi and P. mandapamensis. Blank regions in P. mandapamensis strains indicate that the sequence is not available; dashed lines in P. mandapamensis ATCC 27561^T indicate regions where the DNA was amplified in this study but not sequenced. The hashed rectangles indicate ORFs (orf3 and orf4) flanking the lux-rib₂ operon of fosmid C30-24 that are apparently homologous to bacterial transposases (see the text). In lux-rib₂ of P. leiognathi lelon.2.1, a dashed vertical line in the region homologous to luxC indicates the site of a single nucleotide deletion that causes a frameshift of luxC in this strain, resulting in a stop codon 18 codons later. See the supplemental material for complete maps of the fosmids.

FIG. 2.

PFGE analysis of the chromosomal locations of lux-rib₁ and lux-rib₂. (A) NotI digestion of lnuch.13.1 genomic DNA. (B) PCR amplification using lux-rib₁-specific primers (luxDfor1 and luxArevsec#3) (see http://www-personal.umich.edu/∼pvdunlap/supplementalinfo.html) with genomic fragments NotI-a through NotI-g as templates. (C) PCR amplification using lux-rib₂-specific primers (luxDfor1 and luxArevprim#3) (see http://www-personal.umich.edu/∼pvdunlap/supplementalinfo.html) and genomic fragments NotI-a through NotI-g as templates. Genomic DNA of lnuch.13.1 was used as a positive control (data not shown).

FIG. 3.

Phylogenetic hypothesis of relationships among luminous bacteria based on lux and rib gene sequences. The tree is unrooted because bacterial lux genes are present only in members of the Vibrionaceae, Shewanellaceae, and Enterobacteriaceae; no outgroup bearing the lux genes is known. The total number of aligned nucleotide positions in the data set is 11,021; exclusion of noncoding spacer regions and parsimony-uninformative characters resulted in 4,745 nucleotides for analysis. The single most parsimonious hypothesis is shown (length, 13,739; consistency index, 0.615; retention index, 0.712). Numbers at nodes are jackknife resampling values. The two lux-rib operons from P. leiognathi strains lnuch.13.1, lelon.2.1, and lnuch.21.1 (circled with dashed line) have distinct sequences but are each other's closest relatives (circled inset below the main figure; the asterisk indicates that the jackknife value is for the same branch on each part of the figure). The primary lux-rib operon (lux-rib₁) is proximal to putA in all strains, whereas the secondary lux-rib operon (lux-rib₂) is flanked by putative bacterial transposases in strain lnuch.13.1. Phylogenetic analysis based on housekeeping genes (i.e., gapA, gyrB, recA, rpoA, and rpoD) of Photobacterium species yielded trees consistent with the lux-rib hypothesis shown here.

FIG. 4.

Alignment of a region of luxB from lux-rib₁ and lux-rib₂ of P. leiognathi lnuch.21.1. (A) Nucleotide alignment. (B) Amino acid alignment. Asterisks highlight differences between the sequences; variable nucleotides are shown in boldface type.

FIG. 5.

Map of Japan and Southeast Asia showing the geographic origins of P. leiognathi strains bearing single or multiple lux-rib operons. The scale bar is approximately 500 km. The inset shows an enlarged map of the main islands of Japan (some landmasses were omitted for clarity). Locations: a, Sagami Bay, Kanagawa Prefecture, Honshu, Japan; b, Suruga Bay, Shizuoka Prefecture, Honshu, Japan; c, Wakasa Bay, Fukui Prefecture, Honshu, Japan; d, Ago Bay, Mie Prefecture, Honshu, Japan; e, Tosa Bay, Kochi Prefecture, Shikoku, Japan; f, Nakagusuku Bay, Okinawa-honto, Okinawa Prefecture, Japan; g, Funauki Bay, Iriomote Island, Okinawa Prefecture, Japan; h, Taipei, Taiwan; i, Dahsi, Taiwan; j, Manila Bay, Luzon, Philippine Islands; k, Iloilo, Panay, Philippine Islands; l, Palawan, Philippine Islands; m, Gulf of Thailand. Numbers next to each location indicate the number of strains identified as bearing single (white area in circle) or multiple (gray area in circle) lux-rib operons.