Genome organization and localization of the pufLM genes of the photosynthesis reaction center in phylogenetically diverse marine Alphaproteobacteria

Abstract

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.

FIG. 1.

Phylogenetic tree of Alphaproteobacteria showing the affiliation of facultative anaerobic and aerobic anoxygenic phototrophs (boldface type). Reference organisms are shown in italics; strains investigated here are boxed. EMBL accession numbers are indicated in parentheses. The tree is based on nearly complete 16S rRNA gene sequences and was calculated by using the maximum-likelihood algorithm. Bootstrap values above 50% are indicated. The bar corresponds to 10 base substitutions per 100 nucleotide positions. Flexibacter maritimus (M64629) was used as an out-group.

FIG. 2.

Macrorestriction analysis of the genomic DNA of R. litoralis DSM6996^T hydrolyzed with the endonucleases I-CeuI, PacI, PmeI, and SwaI. Different size ranges of fragments were resolved on various gels, three of which are shown. (A) High size range; (B) medium size range; (C) low size range (see Materials and Methods for details). Restriction fragments are numbered and designated with respect to the endonucleases I-CeuI (Ce), PacI (Pa), PmeI (Pm), and SwaI (Sw). Chromosomes of S. pombe 927 h (S.p.), H. wingei YB-4662-VIA (H.w.), S. cerevisiae YPH80 (S.c.), concatemers of bacteriophage lambda (λ), and a low-range PFGE marker (λlr) were used as size standards.

FIG. 3.

(A) PFGE of undigested HMW genomic DNA of strain DFL-43, strain DFL-11, R. halodurans DSM15395^T (Rs. halo.), R. veldkampii DSM11550^T (Rb. veld.), R. sphaeroides DSM159^T(Rb. sphae), strain DFL-27, strain DFL-12, R. litoralis DSM6996^T (R. lit.), S. guttiformis DSM11458^T (S. gutt.), strain DFL-24, and R. tolerans DSM11457^T (Rv. tol.) inside agarose plugs. λ, low-range PFGE marker; CHR, chromosomal DNA. PFGE parameter set A was a 1% (wt/vol) agarose gel, with pulse times of 0.1 to 25 s for 22.5 h at 6 V/cm and 14°C. (B) Schematic view of panel A. Linear plasmids are represented as bold lines; circular plasmids are represented as dotted lines. Sizes are given above or below the lines.

FIG. 4.

Southern blot hybridization of the DIG-labeled probe pufD12 against PFGE-separated (Fig. 2) and immobilized HMW genomic DNA of strain DFL-43, strain DFL-11, R. halodurans DSM15395^T (Rs. halo.), R. veldkampii DSM11550^T (Rb. veld.), R. sphaeroides DSM159^T(Rb. sphae), strain DFL-27, strain DFL-12, R. litoralis DSM6996^T (R. lit.), S. guttiformis DSM11458^T (S. gutt.), strain DFL-24, and R. tolerans DSM11457^T (Rv. tol.). λ, low-range PFGE marker; CHR, chromosomal DNA.