Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus.

A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells

Impairment of a cyanobacterial glycosyltransferase that modifies a pilin results in biofilm development.

A biofilm inhibiting mechanism operates in the cyanobacterium Synechococcus elongatus. Here, we demonstrate that the glycosyltransferase homologue, Ogt, participates in the inhibitory process - inactivation of ogt results in robust biofilm formation. Furthermore, a mutational approach shows requirement of the glycosyltransferase activity for biofilm inhibition. This enzyme is necessary for glycosylation of the pilus subunit and for adequate pilus formation. In contrast to wild-type culture in which most cells exhibit several pili, only 25% of the mutant cells are piliated, half of which possess a single pilus. In spite of this poor piliation, natural DNA competence was similar to that of wild-type; therefore, we propose that the unglycosylated pili facilitate DNA transformation. Additionally, conditioned medium from wild-type culture, which contains a biofilm inhibiting substance(s), only partially blocks biofilm development by the ogt-mutant. Thus, we suggest that inactivation of ogt affects multiple processes including production or secretion of the inhibitor as well as the ability to sense or respond to it

Collapsing Aged Culture of the Cyanobacterium <i>Synechococcus elongatus</i> Produces Compound(s) Toxic to Photosynthetic Organisms

<div><p>Phytoplankton mortality allows effective nutrient cycling, and thus plays a pivotal role in driving biogeochemical cycles. A growing body of literature demonstrates the involvement of regulated death programs in the abrupt collapse of phytoplankton populations, and particularly implicates processes that exhibit characteristics of metazoan programmed cell death. Here, we report that the cell-free, extracellular fluid (conditioned medium) of a collapsing aged culture of the cyanobacterium <i>Synechococcus elongatus</i> is toxic to exponentially growing cells of this cyanobacterium, as well as to a large variety of photosynthetic organisms, but not to eubacteria. The toxic effect, which is light-dependent, involves oxidative stress, as suggested by damage alleviation by antioxidants, and the very high sensitivity of a catalase-mutant to the conditioned medium. At relatively high cell densities, <i>S. elongatus</i> cells survived the deleterious effect of conditioned medium in a process that required <i>de novo</i> protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in <i>S. elongatus</i> cells, but also resulted in bleaching of pigments in a cell free extract. The latter observation indicates that the elicited damage is a direct effect that does not require an intact cell, and therefore, is mechanistically different from the metazoan-like programmed cell death described for phytoplankton. We suggest that <i>S. elongatus</i> in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program.</p></div

Different fates of aged cultures of the cyanobacterium <i>S. elongatus</i>.

<p>(<b>A</b>) Cultures maintained at stationary phase were characterized by dark blue-green pigmentation up to about 3 months (culture No. 1). Older cultures either rapidly collapsed (culture No. 2), or gradually acquired a yellowish color (culture No. 3) and further survived. Cultures No. 1 and 2 are about 3 months old, whereas culture No. 3 is 6 months old. (<b>B</b>) Conditioned medium (CM) of a collapsing culture induced rapid cell death of exponentially growing cells of <i>S. elongatus</i> in contrast to medium from non-collapsing cultures, which did not affect viability. Cells were inoculated into fresh medium (FM) as a control. 5 µl of undiluted cultures or cultures diluted 1∶100 or 1∶1000 were 'spotted' on solid growth medium, following exposure to the different media (see Methods).</p

Light-dependent toxic effect of conditioned medium from collapsing culture.

<p>Viability assessment using Sytox green (<b>A</b>), and absorbance spectra (<b>B</b>) of exponentially growing cells 24 h after inoculation into fresh growth medium (FM), or into conditioned medium of a collapsing culture (CM). Exposure to CM under light resulted in bleaching of cell pigments including chlorophyll (Chl) and phycocyanin (PC).</p

CM is harmful to photosynthetic microorganisms but not to heterotrophic bacteria.

<p>(<b>A</b>) XAD-extract of CM was applied to variety of cyanobacteria and algae including <i>S. elongatus, Anabaena</i> PCC 7120, <i>Calothrix</i> PCC 7601, <i>Chlamydomonas reinhardtii</i>, <i>Dunaliella salina</i> and <i>Chlorella vulgaris</i>. Sensitivity was examined spectroscopically to detect the effect on pigmentation. Additional phytoplankton species were examined, as well (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100747#pone.0100747.s004" target="_blank">Table S1</a>). Substances extracted with XAD were dissolved in ethanol; this organic solvent was added to fresh medium (FM) in the control samples. (<b>B</b>) <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Streptococcus faecalis</i> and <i>Bacillus cereus</i> were inoculated into FM or CM. The number of colony forming units (CFU) following 12 h illumination in CM was normalized to CFU obtained following exposure to FM.</p

The TD34-mutant of <i>Synechocystis</i>, which lacks a functional photosystem II reaction center, remains sensitive to conditioned medium (CM).

<p>Shown are wild type <i>Synechocystis</i> PCC6803 (6803) and the mutant in which the three copies of <i>psbA</i>, each encoding the D1 protein of photosystem II, were inactivated (TD34). Cells were inoculated into fresh medium (FM) or CM.</p

Cells respond to CM in a density dependent manner.

<p>(<b>A</b>) Exponentially growing cells of <i>S. elongatus</i> were inoculated into conditioned medium (CM) at different initial cell densities, as indicated by the optical density at 750 nm (OD₇₅₀). (<b>B</b>) Chloramphenicol, an inhibitor of protein synthesis, impaired the ability of high density cultures to cope with the deleterious effect of CM. (<b>C</b>) Cells coped better with CM when it was not supplemented with nutrients. (<b>D and E</b>) Inactivation of the <i>nblR</i> gene, encoding a response regulator essential for nutrient starvation responses, results in extreme sensitivity to CM. Data shown in <b>B-D</b> represent cells exposed to CM at OD₇₅₀ = 0.04. Note the different <i>y</i> axis scale in (<b>C</b>) versus (<b>B</b>) and (<b>D</b>).</p