Systematic analysis of nonprogrammed frameshift suppression in E. coli via translational tiling proteomics.

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting. The fact that these programmed events, commonly involving a shift to the -1 frame, occur at specific evolutionarily optimized "slippery" sites has facilitated mechanistic investigation. By contrast, less is known about the scope and nature of error (i.e., nonprogrammed) frameshifting. Here, we examine error frameshifting by monitoring spontaneous frameshift events that suppress the effects of single base pair deletions affecting two unrelated test proteins. To map the precise sites of frameshifting, we developed a targeted mass spectrometry-based method called "translational tiling proteomics" for interrogating the full set of possible -1 slippage events that could produce the observed frameshift suppression. Surprisingly, such events occur at many sites along the transcripts, involving up to one half of the available codons. Only a subset of these resembled canonical "slippery" sites, implicating alternative mechanisms potentially involving noncognate mispairing events. Additionally, the aggregate frequency of these events (ranging from 1 to 10% in our test cases) was higher than we might have anticipated. Our findings point to an unexpected degree of mechanistic diversity among ribosomal frameshifting events and suggest that frameshifted products may contribute more significantly to the proteome than generally assumed.

Role of natural transformation in the evolution of small cryptic plasmids in Synechocystis sp. PCC 6803.

Small cryptic plasmids have no clear effect on the host fitness and their functional repertoire remains obscure. The naturally competent cyanobacterium Synechocystis sp. PCC 6803 harbours several small cryptic plasmids; whether their evolution with this species is supported by horizontal transfer remains understudied. Here, we show that the small cryptic plasmid DNA is transferred in the population exclusively by natural transformation, where the transfer frequency of plasmid-encoded genes is similar to that of chromosome-encoded genes. Establishing a system to follow gene transfer, we compared the transfer frequency of genes encoded in cryptic plasmids pCA2.4 (2378 bp) and pCB2.4 (2345 bp) within and between populations of two Synechocystis sp. PCC 6803 labtypes (termed Kiel and Sevilla). Our results reveal that plasmid gene transfer frequency depends on the recipient labtype. Furthermore, gene transfer via whole plasmid uptake in the Sevilla labtype ranged among the lowest detected transfer rates in our experiments. Our study indicates that horizontal DNA transfer via natural transformation is frequent in the evolution of small cryptic plasmids that reside in naturally competent organisms. Furthermore, we suggest that the contribution of natural transformation to cryptic plasmid persistence in Synechocystis is limited.

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.

Natural Competence in the Filamentous, Heterocystous Cyanobacterium Chlorogloeopsis fritschii PCC 6912.

Lateral gene transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA transfer via natural transformation depends on the ability of recipient cells to actively transport DNA from the environment into the cytoplasm, termed natural competence, which relies on the presence of type IV pili and other competence proteins. Natural competence has been described in cyanobacteria for several organisms, including unicellular and filamentous species. However, natural competence in cyanobacteria that differentiate specialized cells for N2-fixation (heterocysts) and form branching or multiseriate cell filaments (termed subsection V) remains unknown. Here, we show that genes essential for natural competence are conserved in subsection V cyanobacteria. Furthermore, using the replicating plasmid pRL25C, we experimentally demonstrate natural competence in a subsection V organism: Chlorogloeopsis fritschii PCC 6912. Our results suggest that natural competence is a common trait in cyanobacteria forming complex cell filament morphologies. IMPORTANCE Cyanobacteria are crucial players in the global biogeochemical cycles, where they contribute to CO2- and N2-fixation. Their main ecological significance is the primary biomass production owing to oxygenic photosynthesis. Cyanobacteria are a diverse phylum, in which the most complex species differentiate specialized cell types and form true-branching or multiseriate cell filament structures (termed subsection V cyanobacteria). These bacteria are considered a peak in the evolution of prokaryotic multicellularity. Among others, species in that group inhabit fresh and marine water habitats, soil, and extreme habitats such as thermal springs. Here, we show that the core genes required for natural competence are frequent in subsection V cyanobacteria and demonstrate for the first time natural transformation in a member of subsection V. The prevalence of natural competence has implications for the role of DNA acquisition in the genome evolution of cyanobacteria. Furthermore, the presence of mechanisms for natural transformation opens up new possibilities for the genetic modification of subsection V cyanobacteria.

A Bacterial Cell-Based Assay To Study SARS-CoV-2 Protein-Protein Interactions.

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of proteins encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we describe the use of a convenient bacterial cell-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome. We identified 16 distinct intraviral protein-protein interactions (PPIs), involving 16 proteins. We found that many of the identified proteins interact with more than one partner. Further, our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified B2H system that permits the detection of disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm, and we used this system to detect the interaction of the SARS-CoV-2 spike protein receptor-binding domain (RBD) with its cognate cell surface receptor ACE2. We then examined how the RBD-ACE2 interaction is perturbed by several RBD amino acid substitutions found in currently circulating SARS-CoV-2 variants. Our findings illustrate the utility of a genetically tractable bacterial system for probing the interactions of viral proteins and investigating the effects of emerging mutations. In principle, the system could also facilitate the identification of potential therapeutics that disrupt specific interactions of virally encoded proteins. More generally, our findings establish the feasibility of using a B2H system to detect and dissect disulfide bond-dependent interactions of eukaryotic proteins. IMPORTANCE Understanding how virally encoded proteins interact with one another is essential in elucidating basic viral biology, providing a foundation for therapeutic discovery. Here, we describe the use of a versatile bacterial cell-based system to investigate the interactions of the protein set encoded by SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. We identified 16 distinct intraviral protein-protein interactions, involving 16 proteins, many of which interact with more than one partner. Our system facilitates the genetic dissection of these interactions, enabling the identification of selectively disruptive mutations. We also describe a modified version of our bacterial cell-based system that permits detection of the interaction between the SARS-CoV-2 spike protein (specifically, its receptor-binding domain) and its cognate human cell surface receptor ACE2, and we investigated the effects of spike mutations found in currently circulating SARS-CoV-2 variants. Our findings illustrate the general utility of our system for probing the interactions of virally encoded proteins.

Two novel heteropolymer-forming proteins maintain the multicellular shape of the cyanobacterium Anabaena sp. PCC 7120.

Polymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits - for example, MreB and FtsZ in bacteria - or heteropolymers that are composed of two subunits, for example, keratin and α/ß tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament-forming cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) that assemble into a heteropolymer and function in the maintenance of the Anabaena multicellular shape (termed trichome). The two CCRPs - Alr4504 and Alr4505 (named ZicK and ZacK) - are strictly interdependent for the assembly of protein filaments in vivo and polymerize nucleotide independently in vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linear Anabaena trichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize the Anabaena trichome and are likely essential for the manifestation of the multicellular shape in Anabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

Structural Determinants and Their Role in Cyanobacterial Morphogenesis.

Cells have to erect and sustain an organized and dynamically adaptable structure for an efficient mode of operation that allows drastic morphological changes during cell growth and cell division. These manifold tasks are complied by the so-called cytoskeleton and its associated proteins. In bacteria, FtsZ and MreB, the bacterial homologs to tubulin and actin, respectively, as well as coiled-coil-rich proteins of intermediate filament (IF)-like function to fulfil these tasks. Despite generally being characterized as Gram-negative, cyanobacteria have a remarkably thick peptidoglycan layer and possess Gram-positive-specific cell division proteins such as SepF and DivIVA-like proteins, besides Gram-negative and cyanobacterial-specific cell division proteins like MinE, SepI, ZipN (Ftn2) and ZipS (Ftn6). The diversity of cellular morphologies and cell growth strategies in cyanobacteria could therefore be the result of additional unidentified structural determinants such as cytoskeletal proteins. In this article, we review the current advances in the understanding of the cyanobacterial cell shape, cell division and cell growth.

The role of the cytoskeletal proteins MreB and FtsZ in multicellular cyanobacteria.

Multiseriate and true-branching cyanobacteria are at the peak of prokaryotic morphological complexity. However, little is known about the mechanisms governing multiplanar cell division and morphogenesis. Here, we study the function of the prokaryotic cytoskeletal proteins, MreB and FtsZ in Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912. Vancomycin and HADA labeling revealed a mixed apical, septal, and lateral trichome growth mode in F. muscicola, whereas C. fritschii exhibits septal growth. In all morphotypes from both species, MreB forms either linear filaments or filamentous strings and can interact with FtsZ. Furthermore, multiplanar cell division in F. muscicola likely depends on FtsZ dosage. Our results lay the groundwork for future studies on cytoskeletal proteins in morphologically complex cyanobacteria.

A novel septal protein of multicellular heterocystous cyanobacteria is associated with the divisome.

Cyanobacteria are unique among the eubacteria as they possess a hybrid Gram phenotype, having an outer membrane but also a comparably thick peptidoglycan sheet. Furthermore, the cyanobacterial divisome includes proteins specific for both the Gram types as well as cyanobacteria-specific proteins. Cells in multicellular cyanobacteria share a continuous periplasm and their cytoplasms are connected by septal junctions that enable communication between cells in the filament. The localization of septal junction proteins depends on interaction with the divisome, however additional yet unknown proteins may be involved in this process. Here, we characterized Alr3364 (termed SepI), a novel septal protein that interacts with the divisome in the multicellular heterocystous cyanobacterium Anabaena sp. strain PCC 7120. SepI localized to the Z-ring and the intercellular septa but did not interact with FtsZ. Instead, SepI interacted with the divisome proteins ZipN, SepF and FtsI and with the septal protein SepJ. The inactivation of sepI led to a defect in cell filament integrity, colony and cell morphology, septum size, nanopore formation and peptidoglycan biogenesis, and inability to differentiate heterocysts. Our results show that SepI plays a role in intercellular communication and furthermore indicate that SepI functions in the coordination of septal junction localization during cell division.

Identification and characterization of novel filament-forming proteins in cyanobacteria.

Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

Inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. The spike protein of MERS-CoV (MERS-S) facilitates viral entry into host cells, which depends on activation of MERS-S by cellular proteases. Proteolytic activation of MERS-S during viral uptake into target cells has been demonstrated. However, it is unclear whether MERS-S is also cleaved during S protein synthesis in infected cells and whether cleavage is required for MERS-CoV infectivity. Here, we show that MERS-S is processed by proprotein convertases in MERS-S-transfected and MERS-CoV-infected cells and that several RXXR motifs located at the border between the surface and transmembrane subunit of MERS-S are required for efficient proteolysis. However, blockade of proprotein convertases did not impact MERS-S-dependent transduction of target cells expressing high amounts of the viral receptor, DPP4, and did not modulate MERS-CoV infectivity. These results show that MERS-S is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for S protein activation. Efforts to inhibit MERS-CoV infection by targeting host cell proteases should therefore focus on enzymes that process MERS-S during viral uptake into target cells.