Self-suppression of biofilm

Self-suppression of biofilm

Biofilms are consortia of bacteria that are held together by an extracellular matrix. Cyanobacterial biofilms, which are highly ubiquitous and inhabit diverse niches, are often associated with biological fouling and cause severe economic loss. Information on the molecular mechanisms underlying biofilm formation in cyanobacteria is scarce. We identified a mutant of the cyanobacterium Synechococcus elongatus, which unlike the wild type, developed biofilms. This biofilm-forming phenotype is caused by inactivation of homologues of type II secretion /type IV pilus assembly systems and is associated with impairment of protein secretion. The conditioned medium from a wild-type culture represses biofilm formation by the secretion-mutants. This suggested that the planktonic nature of the wild-type strain is a result of a self-suppression mechanism, which depends on the deposition of a factor to the extracellular milieu. The particular niche conditions will determine whether the inhibitor will accumulate to effective levels and thus the described mechanism allows switching to a sessile mode of existence.

selfsup

Left panels: The T2SE-mutant (T2SEΩ) adheres to the growth tube, in contrast to the wild-type strain, which is characterized by planktonic growth. Right panels: Fluorescence microscopy reveals biofilms of T2SEΩ.

env

Environ Microbiol. 2013 Jun;15(6):1786-94.
Self-suppression of biofilm formation in the cyanobacterium Synechococcus elongatus.
Schatz D, Nagar E, Sendersky E, Parnasa R, Zilberman S, Carmeli S, Mastai Y, Shimoni E, Klein E, Yeger O, Reich Z, Schwarz R
http://www.ncbi.nlm.nih.gov/pubmed/?term=Self-suppression+of+biofilm+formation+in+the

 

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Genes essential for biofilm

Genes essential for biofilm

We identified genes that are essential for biofilm formation. These genes include small proteins characterized by a double-glycine secretion motif as well as a component of transport system responsible for secretion and maturation of these small proteins.

genes

Sequence of the double-glycine motif of the product of pcc7942_1134 and selected secreted peptides. Cerein7B of Bacillus cereus, EnterocinA and EnterocinB of Enterococcus faceum, MicrocinE492 of Klebsiella pneumonia, and Microcin24, MicrocinH47, and ColicinV of Escherichia coli. Black shading indicates the double-glycine or glycine-alanine present just prior to the peptide cleavage site (arrow).

screp
Scientific Reports –Nature
2016 Aug 25;6:32209
Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.
Parnasa R, Nagar E, Sendersky E, Reich Z, Simkovsky R, Golden S, Schwarz R
http://www.ncbi.nlm.nih.gov/pubmed/27558743

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To be or not to be planktonic?

To be or not to be planktonic?

The transition between planktonic growth and biofilm formation represents a tightly regulated developmental shift that has substantial impact on cell fate. Here, we highlight different mechanisms through which bacteria limit their own biofilm development. The mechanisms involved in these self-inhibition processes include: (i) regulation by secreted small molecules, which govern intricate signalling cascades that eventually decrease biofilm development, (ii) extracellular polysaccharides capable of modifying the physicochemical properties of the substratum and (iii) extracellular DNA that masks an adhesive structure. These mechanisms, which rely on substances produced by the bacterium and released into the extracellular milieu, suggest regulation at the communal level. In addition, we provide specific examples of environmental cues (e.g. blue light or glucose level) that trigger a cellular response reducing biofilm development. All together, we describe a diverse array of mechanisms underlying self-inhibition of biofilm development in different bacteria and discuss possible advantages of these processes.

planc

Biofilm inhibition in S. elongatus by an extracellular inhibitor.

Wild-type cells produce and secrete an inhibitor (red ‘no-entrance’ sign) that suppresses transcription of the genes Synpcc7942_1133 and Synpcc7942_1134. Inactivation of t2sE, encoding a homologue of subunit E of type two secretion systems (T2S), enables biofilm development. The biofilm-forming mutant, T2SEΩ, is most likely impaired in secretion of the inhibitory factor, and therefore expresses ‘biofilm-genes’ at a higher level, and develops biofilms.

env

Environmental Microbiology 2015 May;17(5):1477-86.

To be or not to be planktonic? Self-inhibition of biofilm development.

Nagar E, Schwarz R.

http://www.ncbi.nlm.nih.gov/pubmed/25088599

 

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Regulated pigment degradation

Regulated pigment degradation

The proteolysis adaptor, NblA, initiates protein pigment degradation

Degradation of the cyanobacterial protein pigment complexes, the phycobilisomes, is a central acclimation response that controls light energy capture. The small protein, NblA, is essential for proteolysis of these large complexes, which may reach a molecular mass of up to 4 MDa. Interactions of NblA in vitro supported the suggestion that NblA is a proteolysis adaptor that labels the pigment proteins for degradation. The mode of operation of NblA in situ, however, remained unresolved. Particularly, it was unclear whether NblA interacts with phycobilisome proteins while part of the large complex, or alternatively interaction with NblA, necessitates dissociation of pigment subunits from the assembly. Fluorescence intensity profiles demonstrated the preferential presence of NblA::GFP (green fluorescent protein) at the photosynthetic membranes, indicating co-localization with phycobilisomes. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for interaction of NblA with phycobilisome protein pigments. Additionally, we demonstrated the role of NblA in vivo as a proteolysis tag based on the rapid degradation of the fusion protein NblA::GFP compared with free GFP. Taken together, these observations demonstrated in vivo the role of NblA as a proteolysis adaptor. Additionally, the interaction of NblA with phycobilisomes indicates that the dissociation of protein pigment subunits from the large complex is not a prerequisite for interaction with this adaptor and, furthermore, implicates NblA in the disassembly of the protein pigment complex. Thus, we suggest that, in the case of proteolysis of the phycobilisome, the adaptor serves a dual function: undermining the complex stability and designating the dissociated pigments for degradation.

 

degradation

Phycobilisome disassembly by NblA.
NblA intercalates into the rod structure, thereby undermining the association between adjacent pigment subunits, resulting in detachment of NblA associated with small pigment assemblies. As rod degradation progresses, the core pigments become accessible to NblA, and the phycobilisome core is disassembled.
Designations: Photosynthetic reaction center II (PSII, green), allophycocyanin (APC, light blue), phycocyanin (PC, blue) and dimers of NblA (orange).

plant1

The Plant Journal 2014 Jul;79(1):118-26.
The proteolysis adaptor, NblA, initiates protein pigment degradation by interacting with the cyanobacterial light-harvesting complexes.
Sendersky E, Kozer N, Levi M, Garini Y, Shav-Tal Y, Schwarz R.
http://www.ncbi.nlm.nih.gov/pubmed/24798071

NblA is essential for degradation of the core pigment

The cyanobacterial light-harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light-harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod-shaped pigment assemblies emanating from the core. NblA, a low-molecular-weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatus PCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.

plant1

The Plant Journal 2015 Sep;83(5):845-52.

The proteolysis adaptor, NblA, is essential for degradation of the core pigment of the cyanobacterial light-harvesting complex.
Sendersky E, Kozer N, Levi M, Moizik M, Garini Y, Shav-Tal Y, Schwarz R.
http://www.ncbi.nlm.nih.gov/pubmed/26173720

NblC, a novel component required for pigment degradation during starvation

Adjustment of photosynthetic light harvesting to ambient conditions is essential to allow efficient energy capturing and to prevent surplus excitation and the cellular damage resulting from it. Degradation of the cyanobacterial light harvesting complex, the phycobilisome, is a general acclimation response occurring under various stress conditions. This study identifies a novel component, NblC, which mediates phycobilisome degradation under nitrogen, sulphur and phosphorus starvation. Our study indicates the requirement of NblC for efficient expression of nblA, an essential component of the degradation pathway; accumulation of nblA transcripts upon nutrient starvation was impaired in the NblC-mutant. Furthermore, expression of NblC under the control of a foreign promoter resulted in accumulation of nblA transcripts and degradation of the light harvesting complex. Transcription of nblC is induced upon nutrient starvation, suggesting the requirement of elevated levels of NblC under these conditions. Importantly, NblC could not exert its positive effect on nblA expression in the absence of the response regulator NblR. Sequence alignment suggests kinase motifs as well as homology of NblC to anti-sigma factors. Accordingly, we suggest a mode of action for this newly identified modulator, which provides new insights into regulation of gene expression in response to environmental stimuli.

molec

Molecular Microbiology 2005 Nov;58(3):659-68.
NblC, a novel component required for pigment degradation during starvation in Synechococcus PCC 7942.
Sendersky E, Lahmi R, Shaltiel J, Perelman A, Schwarz R.
http://www.ncbi.nlm.nih.gov/pubmed/?term=NblC%2C+a+novel+component+required+for+pigment

 

 

Structure-function analysis of NblA

Structure-function analysis of NblA

Structural, functional, and mutational analysis of the NblA protein

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (approximately 6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via “structural mimicry” due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.

struc

NblA crystal structure and positions of mutations resulting in a “non-bleaching phenotype” of S. elongatus.

jbc

The Journal of Biological Chemistry  2008 Oct 31;283(44):30330-40.
Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome.
Dines M, Sendersky E, David L, Schwarz R, Adir N.
http://www.ncbi.nlm.nih.gov/pubmed/18718907


 

 

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Aging cultures produce toxins

Aging cultures produce toxins

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 Synechococcus elongatus 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, S. elongatus cells survived the deleterious effect of conditioned medium in a process that required de novo protein synthesis. Application of conditioned medium from a collapsing culture caused severe pigment bleaching not only in S. elongatus 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 S. elongatus in aged cultures are triggered to produce a toxic compound, and thus, this process may be envisaged as a novel regulated death program.

toxic

Flow cytometric analysis following ‘live-dead staining’ indicates substantial death of cells inoculated into conditioned medium (CM) compared to cells growing in fresh medium (FM).

 

plos1

PLoS One. 2014 Jun 24;9(6):e100747.
Collapsing aged culture of the cyanobacterium Synechococcus elongatus produces compound(s) toxic to photosynthetic organisms.
Cohen A, Sendersky E, Carmeli S, Schwarz R.
http://www.ncbi.nlm.nih.gov/pubmed/24959874

 

 

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Nutrient-dictated cell elongation

Nutrient-dictated cell elongation

While tightly regulated, bacterial cell morphology may change substantially in response to environmental cues. Here we describe such changes in the cyanobacterium Synechococcus sp. strain PCC7942. Once maintained in stationary phase, these rod-shaped organisms stop dividing and elongate up to 50-fold. Increase in cell length of a thymidine-auxotroph strain upon thymidine starvation implies that inhibition of DNA replication underlies cell elongation. Elongation occurs under conditions of limiting phosphorus but sufficient nitrogen levels. Once proliferative conditions are restored, elongated cells divide asymmetrically instead of exhibiting the typical fission characterized by mid-cell constriction. The progeny are of length characteristic of exponentially growing cells and are proficient of further proliferation. We propose that the ability to elongate under conditions of cytokinesis arrest together with the rapid induction of cell division upon nutrient repletion represents a beneficial cellular mechanism operating under specific nutritional conditions.

nutriendict

Cell elongation upon culture aging.
A. Distribution of cell length at various times following inoculation. Data points represent the number of cells falling within bins of 0.5mm; trend lines are shown for clarity. Exp. – exponential growth phase (2-day-old cultures).
B. Median cell length as a function of time.
C. An image of elongated (8-week-old) cells. Scale bar: 10mm.

env 

Environ Microbiol. 2012 Mar;14(3):680-90.
Nutrient-associated elongation and asymmetric division of the cyanobacterium Synechococcus PCC 7942.
Goclaw-Binder H, Sendersky E, Shimoni E, Kiss V, Reich Z, Perelman A, Schwarz R.
Link: http://www.ncbi.nlm.nih.gov/pubmed/22026402

 

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Tailoring cyanobacteria for biofuel

Tailoring cyanobacteria for biofuel

Environmental as well as economic reasons call for a sustainable alternative for the use of fossil fuels. Light energy harnessed by photosynthetic organisms provides an attractive mean for biofuel production. Biofuels derived from sugars and oils found in arable crops have already been commercialized; however, these processes are not cost-effective. Additionally, the impact of these ‘first generation biofuels’ on food supply and price have raised ethical questions and encouraged a search for an alternative biomass source. Cyanobacteria offer an efficient mean for biofuel production that is not associated with the current problems of land-based-biofuel feedstock. They combine the benefits of fast growing simple microorganisms with a cost effective mode of growth based on light energy and mineral nutrients. We aim at establishing a simple process for bioethanol by yeast fermentation. We modify cyanobacteria to increase glycogen level as raw material for bioethanol. (Manuscript in preparation).

tail

Schematic presentation of glycogen metabolism.
ADP-glucose pyrophosphorylase (GlgC) catalyzes the first committed step in glycogen synthesis by converting glucose-1- phosphate (Glc-1-P) to ADP-Glucose (ADPG).  The next two sequential steps are catalyzed by glycogen synthase (GlgA), and branching enzyme (GlgB), respectively. Carbon skeletons are fed into this pathway, initially by the activity of Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) that catalyzes the CO2 fixation step of the Calvin-Benson-Bassham (CBB) cycle. The RuBisCO product, 3-phosphoglycerate (PGA), is stepwise converted into glyceraldehyde 3-phosphate (GAP) and further on by gluconeogenic enzymes (marked by an asterisk) to glucose and Glc-1-P. Glycogen catabolism is mediated by glycogen phosphorylase (GlgP) and debranching enzymes (The cyanobacterium used in the proposed study, Synechococcus elongatus possesses two homologs of debranching enzymes annotated, GlgX and DBE).  (This scheme is modified from Wilson et al. FEMS Microbiol Rev, 2010. 34(6): p. 952-85.)

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