Natural competence in Chlorogloeopsis fritschii PCC 6912 and other ramified cyanobacteria

Lateral DNA transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the environment. The ability of recipient cells to actively transport DNA into the cytoplasm – termed natural competence – depends on the presence of type IV pili and competence proteins. Natural competence has been described in cyanobacteria for several organisms including unicellular and filamentous species. However, the presence of natural competence in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes essential for natural competence and experimentally demonstrate natural competence in the ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). Searching for homologs to known natural competence genes in ramified cyanobacteria showed that these genes are conserved in the majority of tested isolates. Experimental validation of natural competence using several alternative protocols demonstrates that Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show that natural competence is a common trait in ramified cyanobacteria and that natural transformation is likely to play an important role in cyanobacteria evolution.ImportanceCyanobacteria are crucial players in the global biogeochemical cycles where they contribute to CO2- and N2-fixation. Their main ecological significance is the oxygen-producing photosynthetic apparatus that contributes to contemporary food chains. Ramified cyanobacteria form true-branching and multiseriate cell filament structures that represent a peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. Here we show that ramified cyanobacteria harbor the mechanisms required for DNA acquisition via natural transformation. The prevalence of mechanisms for natural uptake of DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria opens up new possibilities for genetic modification of ramified cyanobacteria.

Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27

ABSTRACT Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.

Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in Acinetobacter sp. Strain BD413 andThermus thermophilus HB27

ABSTRACT The mesophile Acinetobacter sp. strain BD413 and the extreme thermophile Thermus thermophilus HB27 display high frequencies of natural transformation. In this study we identified and characterized a novel competence gene in Acinetobacter sp. strain BD413, comA, whose product displays significant similarities to the competence proteins ComA and ComEC inNeisseria and Bacillus species. Transcription of comA correlated with growth phase-dependent transcriptional regulation of the recently identified pilin-like factors of the transformation machinery. This finding strongly suggests that comA is part of a competence regulon. Examination of the genome sequence of T. thermophilus HB27 led to detection of a comA/comEC-like open reading frame (ORF) which is flanked by an ORF whose product shows significant similarities to the Bacillus subtilis competence protein ComEA. To examine whether these two ORFs, designated comEC andcomEA, are implicated in natural transformation of T. thermophilus HB27, both were disrupted by using a thermostable kanamycin resistance marker. Natural transformation in comEC mutants was reduced 1,000-fold, whereas in comEA mutants the natural transformation phenotype was completely eliminated. These results strongly suggest that both genes, comEC and comEA, are required for natural transformation in T. thermophilus HB27. Several transmembrane α-helices are predicted based on the amino acid sequences of ComA in Acinetobacter sp. strain BD413 and ComEC in T. thermophilus HB27, which suggests that ComA and ComEC are located in the inner membrane and function in DNA transport through the cytoplasmic membrane.