SepT, a novel protein specific to multicellular cyanobacteria, influences peptidoglycan growth and septal nanopore formation in Anabaena sp. PCC 7120.

IMPORTANCE: Multicellular organization is a requirement for the development of complex organisms, and filamentous cyanobacteria such as Anabaena represent a paradigmatic case of bacterial multicellularity. The Anabaena filament can include hundreds of communicated cells that exchange nutrients and regulators and, depending on environmental conditions, can include different cell types specialized in distinct biological functions. Hence, the specific features of the Anabaena filament and how they are propagated during cell division represent outstanding biological issues. Here, we studied SepT, a novel coiled-coil-rich protein of Anabaena that is located in the intercellular septa and influences the formation of the septal specialized structures that allow communication between neighboring cells along the filament, a fundamental trait for the performance of Anabaena as a multicellular organism.

SepN is a septal junction component required for gated cell-cell communication in the filamentous cyanobacterium Nostoc.

Multicellular organisms require controlled intercellular communication for their survival. Strains of the filamentous cyanobacterium Nostoc regulate cell-cell communication between sister cells via a conformational change in septal junctions. These multi-protein cell junctions consist of a septum spanning tube with a membrane-embedded plug at both ends, and a cap covering the plug on the cytoplasmic side. The identities of septal junction components are unknown, with exception of the protein FraD. Here, we identify and characterize a FraD-interacting protein, SepN, as the second component of septal junctions in Nostoc. We use cryo-electron tomography of cryo-focused ion beam-thinned cyanobacterial filaments to show that septal junctions in a sepN mutant lack a plug module and display an aberrant cap. The sepN mutant exhibits highly reduced cell-cell communication rates, as shown by fluorescence recovery after photobleaching experiments. Furthermore, the mutant is unable to gate molecule exchange through septal junctions and displays reduced filament survival after stress. Our data demonstrate the importance of controlling molecular diffusion between cells to ensure the survival of a multicellular organism.

Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria.

Contractile injection systems (CISs) mediate cell-cell interactions by phage tail-like structures, using two distinct modes of action: extracellular CISs are released into the medium, while type 6 secretion systems (T6SSs) are attached to the cytoplasmic membrane and function upon cell-cell contact. Here, we characterized a CIS in the multicellular cyanobacterium Anabaena, with features distinct from extracellular CISs and T6SSs. Cryo-electron tomography of focused ion beam-milled cells revealed that CISs were anchored in thylakoid membrane stacks, facing the cell periphery. Single particle cryo-electron microscopy showed that this unique in situ localization was mediated by extensions of tail fibre and baseplate components. On stress, cyanobacteria induced the formation of ghost cells, presenting thylakoid-anchored CISs to the environment. Functional assays suggest that these CISs may mediate ghost cell formation and/or interactions of ghost cells with other organisms. Collectively, these data provide a framework for understanding the evolutionary re-engineering of CISs and potential roles of these CISs in cyanobacterial programmed cell death.

Plasmid-Chromosome Crosstalk in Staphylococcus aureus: A Horizontally Acquired Transcription Regulator Controls Polysaccharide Intercellular Adhesin-Mediated Biofilm Formation.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.

Cell-cell communication through septal junctions in filamentous cyanobacteria.

Septal junctions are cell-cell connections that mediate intercellular communication in filamentous cyanobacteria. The septal peptidoglycan is perforated by dozens of 20 nm-wide nanopores, through which these proteinaceous structures traverse, physically connecting adjacent cells. On each cytoplasmic side, every septal junction contains a flexible cap structure that closes the connection in a reversible manner upon stress. This gating mechanism reminds of the gap junctions from metazoans and represents a primordial control system for cell-cell communication. In this review, we summarize the knowledge about formation of the nanopore array as the framework for incorporation of cell-cell connecting septal junctions. Furthermore, the architecture of septal junctions, proteins involved in septal junction constitution and regulation of intercellular communication will be addressed.

A nanopore array in the septal peptidoglycan hosts gated septal junctions for cell-cell communication in multicellular cyanobacteria.

Some filamentous cyanobacteria are phototrophic bacteria with a true multicellular life style. They show patterned cell differentiation with the distribution of metabolic tasks between different cell types. This life style requires a system of cell-cell communication and metabolite exchange along the filament. During our study of the cell wall of species Nostoc punctiforme and Anabaena sp. PCC 7120 we discovered regular perforations in the septum between neighboring cells, which we called nanopore array. AmiC-like amidases are drilling the nanopores with a diameter of 20 nm, and are essential for communication and cell differentiation. NlpD-like regulators of AmiC activity and septum localized proteins SepJ, FraC and FraD are also involved in correct nanopore formation. By focused ion beam (FIB) milling and electron cryotomography we could visualize the septal junctions, which connect adjacent cells and pass thru the nanopores. They consist of cytoplasmic caps, which are missing in the fraD mutant, a plug inside the cytoplasmic membrane and a tube like conduit. A destroyed membrane potential and other stress factors lead to a conformational change in the cap structure and loss of cell-cell communication. These gated septal junctions of cyanobacteria are ancient structures that represent an example of convergent evolution, predating metazoan gap junctions.

Structure and Function of a Bacterial Gap Junction Analog.

Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.

Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae.

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.