Natural Competence in the Filamentous, Heterocystous Cyanobacterium Chlorogloeopsis fritschii PCC 6912

Abstract

Lateral gene transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA transfer via natural transformation depends on the ability of recipient cells to actively transport DNA from the environment into the cytoplasm, termed natural competence, which relies on the presence of type IV pili and other competence proteins. Natural competence has been described in cyanobacteria for several organisms, including unicellular and filamentous species. However, natural competence in cyanobacteria that differentiate specialized cells for N₂-fixation (heterocysts) and form branching or multiseriate cell filaments (termed subsection V) remains unknown. Here, we show that genes essential for natural competence are conserved in subsection V cyanobacteria. Furthermore, using the replicating plasmid pRL25C, we experimentally demonstrate natural competence in a subsection V organism: Chlorogloeopsis fritschii PCC 6912. Our results suggest that natural competence is a common trait in cyanobacteria forming complex cell filament morphologies. IMPORTANCE Cyanobacteria are crucial players in the global biogeochemical cycles, where they contribute to CO₂- and N₂-fixation. Their main ecological significance is the primary biomass production owing to oxygenic photosynthesis. Cyanobacteria are a diverse phylum, in which the most complex species differentiate specialized cell types and form true-branching or multiseriate cell filament structures (termed subsection V cyanobacteria). These bacteria are considered a peak in the evolution of prokaryotic multicellularity. Among others, species in that group inhabit fresh and marine water habitats, soil, and extreme habitats such as thermal springs. Here, we show that the core genes required for natural competence are frequent in subsection V cyanobacteria and demonstrate for the first time natural transformation in a member of subsection V. The prevalence of natural competence has implications for the role of DNA acquisition in the genome evolution of cyanobacteria. Furthermore, the presence of mechanisms for natural transformation opens up new possibilities for the genetic modification of subsection V cyanobacteria.

Keywords: cyanobacteria; genome evolution; lateral gene transfer; natural transformation.

FIG 1

Distribution of homologs of the core natural competence genes in subsection V cyanobacteria. Cells in the matrix are colored according to the percent sequence similarity (see color-bar on the right) between the Synechocystis sp. PCC 6803 query protein sequence and the respective subsection V cyanobacterium homologs (strain name shown on the left). The assembly level of the subsection V cyanobacteria genome is given in the left column. Gray cells correspond to missing hits on the protein level, but by tBLASTn, similar sequences can be found (>70% query coverage) on the genomes. Missing annotation of these proteins can correspond to deleterious single nucleotide polymorphisms (SNPs). These SNPs can either have biological relevance or be caused by suboptimal sequencing quality. For the black cell, no specific hits were found by tBLASTn (see Data Set S1 for further information on sequence similarity and additional strains). The level of protein sequence conservation toward the Synechocystis sp. PCC 6803 query proteins differs among the investigated genes. PilT, an ATPase responsible for pilus retraction, is nearly as conserved as RecA, a protein considered to be nearly ubiquitous in bacteria (38). Also, PilB, the antagonistic ATPase for pilus elongation, as well as the pilus components PilC and PilM, the prepilin peptidase PilD, and DprA, Hfq, and EbsA all share a sequence similarity of around 50% or higher with the query protein sequence. The sequences of other parts of the pilus and the competence proteins ComEA, ComEC, and ComF are less conserved.

FIG 2

Natural transformation of Chlorogloeopsis fritschii PCC 6912. (A) Selection of Chlorogloeopsis colonies on nitrocellulose filters. A representative image showing either no transformants on selective BG11 plates after transformation without DNA or 20 ng of pRL25C (left) or Nm-resistant transformants after transformation with larger amounts of pRL25C (right). (B) Transformation frequencies of Chlorogloeopsis with plasmid pRL25C. Transformation frequency is given as the ratio of CFU with antibiotic selection (indicating transformation) to the number of CFU without antibiotic selection. The plot shows the dependency of the transformation frequency on the amount of introduced plasmid pRL25C (0 ng, 20 ng, 200 ng, 2 μg, or 20 μg) and the availability of combined nitrogen before selection (BG11 with 1.5 g/L NaNO₃ and BG11₀ without nitrate). Whiskers show one standard error of the mean. Single transformation frequencies from three independent experiments are given as single data points (red, blue, black).