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

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

Anabaena sp. PCC 7120 grows by forming filaments of communicating cells and is considered a paradigm of bacterial multicellularity. Molecular exchanges between contiguous cells in the filament take place through multiprotein channels that traverse the septal peptidoglycan through nanopores connecting their cytoplasms. Besides, the septal-junction complexes contribute to strengthen the filament. In search for proteins with coiled-coil domains that could provide for cytoskeletal functions in Anabaena, we identified SepT (All2460). SepT is characteristic of the phylogenetic clade of filamentous cyanobacteria with the ability to undergo cell differentiation. SepT-GFP fusions indicate that the protein is located at the cell periphery and, conspicuously, in the intercellular septa. During cell division, the protein is found at midcell resembling the position of the divisome. The bacterial adenylate cyclase two-hybrid analysis shows SepT interactions with itself and putative elongasome (MreB, RodA), divisome (FtsW, SepF, ZipN), and septal-junction (SepJ)-related proteins. Thus, SepT appears to rely on the divisome for localization at mature intercellular septa to form part of intercellular protein complexes. Two independently obtained mutants lacking SepT showed alterations in cell size and impaired septal and peripheral peptidoglycan incorporation during cell growth and division. Notably, both mutants showed conspicuous alterations in the array of nanopores present in the intercellular peptidoglycan disks, including aberrant nanopore morphology, number, and distribution. SepT appears, therefore, to be involved in the control of peptidoglycan growth and the formation of cell-cell communication structures that are at the basis of the multicellular character of this group of cyanobacteria

Identification and characterization of the cell-cell communication system of a multicellular cyanobacterium

Around 2.5 billion years ago, cyanobacteria built the basis for life on Earth by raising the atmospheric oxygen level. Multicellularity among Cyanobacteria evolved very early and increased their fitness due to improved motility, economies of scale and faster adaption to changing environmental conditions. The importance of multicellularity for evolution of life and complex life styles gives reason for performing research on these organisms. Of particular interest is the cell-cell communication system, which allows molecular exchange between cells of the organism via direct cell contact. Without such a system, a multicellular lifestyle with division of labor between specialized cells would be impossible. The cell-cell communication system of the model organism Anabaena sp. PCC 7120 comprises a functional unit of a nanopore array in the septal peptidoglycan and of proteinaceous septal junctions (SJs). The SJs traverse the nanopores to connect the cytoplasm of adjacent cells. Little was known about the mode of action of this communication system and the structural protein components were obscure. This work revealed the in situ architecture of SJs as three-modular complexes involving a cytoplasmic cap and a membrane-embedded plug module at both sites of a length-variable tube module. Furthermore, it could be demonstrated that SJs are gated channels whose caps undergo a structural rearrangement to switch from an open into a closed state, which does not allow cell-to-cell diffusion. Regulation of intercellular diffusion was observed as a response on various intracellular stress conditions. Identification of the septum-localized membrane protein FraD as the first structural SJ component allowed the development of a co-immunoprecipitation using FraD as bait with the aim to discover further proteins of these complexes. By this, a so far hypothetical protein was identified as potential interaction partner of FraD and termed SepN. SJs of a mutant in this septal membrane protein lacked the plug module and exhibited a cap reminiscent to the closed state. Therefore, the function of the SJ plug was assumed to be important for holding the cap structure in its opened position as well as for SJ closure. A mutant in another identified protein, FraI, showed a severely reduced communication, which was linked to a drastic reduction in the number of nanopores. A relation to amidases that drill the nanopore array was therefore suggested. In this work, the complex network of proteins involved in cell-cell communication and the understanding of regulation of intercellular exchange were greatly extended. This includes the identification of two proteins that are essential to form the SJ complex, as well as the establishment of a microscopic assay to screen future mutants for their ability to regulate their communication, and the development of a workflow to identify further (membranous) SJ proteins.Dissertation ist gesperrt bis zum 09.12.2023