The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation

Abstract

The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation The gene slr0388 was previously annotated to encode a hypothetical protein in Synechocystis sp. strain PCC 6803. When a positively phototactic strain of this cyanobacterium was insertionally inactivated at slr0388, the mutants were not transformable, and appeared to aggregate as a result of increased bundling of type IV pili. Also, these mutants were rendered non-phototactic compared to the wild-type. Quantitative real-time PCR revealed a 3?5-fold increase in pilA1 transcript levels in the mutant over wild-type cells, while there were no changes in the level of pilT1 and comA transcripts. Supernatant from mutant liquid culture contained more PilA1 protein, confirmed by mass spectrometric analysis, compared to the wild-type cells, which corresponded to the increase in pilA1 transcripts. The increase in PilA1 subunits may contribute to the bundling morphology of pili that was observed, which in turn may act to retard DNA uptake by hindering the retraction of pili. This gene is therefore proposed to be designated comF, as it possesses a phosphoribosyltransferase domain, a distinguishing feature of other ComF proteins of naturally transformable heterotrophic bacteria. This report is the second of a competence-related gene from Synechocystis sp. strain PCC 6803, the product of which does not show homology to other well-studied type IV pili proteins.

Functional Analysis of PilT from the Toxic Cyanobacterium Microcystis aeruginosa PCC 7806

“…This construct was then introduced into Synechocystis sp. strain PCC 6803 pilT1 mutants via electroporation, as previously described (29), for integration of the M. aeruginosa PCC 7806 pilT gene into the genome of Synechocystis sp. strain PCC 6803 at the locus slr0168.…”

“…Exponentially growing wild-type and mutant Synechocystis sp. strain PCC 6803 cells were transformed with 600 ng of p0769Sp, a vector in which the gene slr0769 is disrupted by a spectinomycin cassette (29). Assays were performed in triplicate, and colonies were counted after 14 to 21 days of growth.…”

“…Motility assays for Synechocystis sp. strain PCC 6803 and P. aeruginosa were performed as described previously (2,29). Competency assays were performed according to the natural transformation protocol described above.…”

“…Synechocystis sp. strain PCC 6803 was naturally transformed with constructed vectors as described elsewhere (29). Competent cells of P. aeruginosa were prepared and transformed as previously described (2).…”

“…For analysis of transcripts from M. aeruginosa PCC 7806, primers designed previously (28) were used to amplify a 100-bp region of the pilA cDNA, a 231-bp region of the pilB cDNA, a 226-bp region of the pilC cDNA, and a 100-bp region of the pilT cDNA. Reverse transcription-PCR of RNA was performed as previously described (29), but with 300 ng of starting total RNA template. Random nonamers (Sigma) were used for cDNA synthesis from M. aeruginosa PCC 7806 RNA.…”