Detection of Prochlorothrix in brackish waters by specific amplification of pcb genes

Abstract

Prochlorothrix hollandica is the only filamentous chlorophyll b (Chlb)-containing oxyphotobacterium that has been found in freshwater habitats to date. Chlb serves as a light-harvesting pigment which is bound to special binding proteins (Pcb). Even though Prochlorothrix was initially characterized as a highly salt-sensitive species, we detected it in a brackish water environment that is characterized by salinities of up to 12 practical salinity units. Using PCR and reverse transcription, we amplified pcb gene fragments of phytoplankton samples taken along a salinity gradient in the eutrophic Darss-Zingst estuary (southern Baltic Sea). After sequencing, high levels of homology to the pcbB and pcbC genes of P. hollandica were found. Furthermore, autofluorescence of Prochlorothrix-like filaments that indicated that Chlb was present was detected in enrichment cultures prepared from the estuarine phytoplankton. The detection of Chlb-containing filaments, as well as the pcb and 16S ribosomal DNA sequences, suggests that Prochlorothrix is an indigenous genus in the Darss-Zingst estuary and may also inhabit many other brackish water environments. The potential of using pcb gene detection to differentiate Prochlorothrix from morphologically indistinguishable species belonging to the genera Pseudanabaena and Planktothrix (Oscillatoria) in phytoplankton analyses is discussed.

FIG. 1.

Map of sampling sites in the Darss-Zingst estuary. Sampling sites 1 to 10 correspond to buoys Recknitz, R2, R84, R37/44, R1/B65, B53, B37/46, B12/13, B18/19, and B35, respectively. The salinity at each sampling site (in PSU) is indicated in parentheses. The inset is a map of the Baltic Sea.

FIG. 2.

Separation of PCR fragments obtained with DNA (A to C) or cDNA (D and E) from 10 sampling sites. The lane numbers indicate the sampling sites (Fig. 1). The following different primer pairs were used on the DNA level: degenerate cyanobacterial 16S rDNA primers CYA359F and CYA781R (A), Prochlorothrix-specific 16S rDNA primers 16S-Pholl-fw and 16S-Pholl-rev (B), and Prochlorothrix-specific pcbC primers pcbCfw and pcbCrev (C). On the cDNA level, degenerate primers optisi-fw, hlwha-l-rev, and pyfadt-rev for pcbB amplification in nested PCR (D) and Prochlorothrix-specific pcbC primers pcbCfw and pcbCrev (E) were used. DNA from cultures of P. hollandica (P.h.) and P. scandica (P.s.) served as positive controls. Lanes M contained a marker (EcoRI/HindIII-digested λ DNA).

FIG. 3.

Rooted cladograms obtained after phylogenetic analyses by the maximum-parsimony method (performed with PAUP, beta version 4.0; Laboratory of Molecular Systematics, Smithsonian Institution) of a 527-bp fragment of the 16S rDNA sequences (maximum-parsimony method) (A) and of a 828-bp fragment of the pcbB and pcbC sequences (neighbor-joining method) (B). Organisms whose sequences were obtained from databases are indicated by asterisks. Selected bootstrap values based on 1,000 replications are shown at the nodes; only values greater than 50% are shown. The cladograms were constructed with TreeView (version 1.5; R. D. M. Page).

FIG. 4.

Relative biomasses of Planktothrix-like filaments in different fractions of the phytoplankton in samples from the Darss-Zingst estuary. The percentages of biomass were calculated for Oscillatoriales (open bars), total cyanobacteria (grey bars), and total phytoplankton (solid bars).

FIG. 5.

Micrographs of cyanobacteria and eukaryotic algae in enrichment cultures from the Darss-Zingst estuary after 14 days of incubation. (A) Bright-field microscopy. (B and C) Epifluorescence microscopy of autofluorescence with illumination that preferentially excited Chla (B) and Chlb (C).