Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Probing the biogenesis pathway and dynamics of thylakoid membranes.

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

The Role of the Cyanobacterial Type IV Pilus Machinery in Finding and Maintaining a Favourable Environment.

Type IV pili (T4P) are proteinaceous filaments found on the cell surface of many prokaryotic organisms and convey twitching motility through their extension/retraction cycles, moving cells across surfaces. In cyanobacteria, twitching motility is the sole mode of motility properly characterised to date and is the means by which cells perform phototaxis, the movement towards and away from directional light sources. The wavelength and intensity of the light source determine the direction of movement and, sometimes in concert with nutrient conditions, act as signals for some cyanobacteria to form mucoid multicellular assemblages. Formation of such aggregates or flocs represents an acclimation strategy to unfavourable environmental conditions and stresses, such as harmful light conditions or predation. T4P are also involved in natural transformation by exogenous DNA, secretion processes, and in cellular adaptation and survival strategies, further cementing the role of cell surface appendages. In this way, cyanobacteria are finely tuned by external stimuli to either escape unfavourable environmental conditions via phototaxis, exchange genetic material, and to modify their surroundings to fit their needs by forming multicellular assemblies.

Factors Controlling Floc Formation and Structure in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Motile strains of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 readily aggregate into flocs, or floating multicellular assemblages, when grown in liquid culture. As described here, we used confocal imaging to probe the structure of these flocs, and we developed a quantitative assay for floc formation based on fluorescence imaging of 6-well plates. The flocs are formed from strands of linked cells, sometimes packed into dense clusters but also containing voids with very few cells. Cells within the dense clusters show signs of nutrient stress, as judged by the subcellular distribution of green fluorescent protein (GFP)-tagged Vipp1 protein. We analyzed the effects on flocculation of a series of mutations that alter piliation and motility, including Δhfq, ΔpilB1, ΔpilT1, and ΔushA mutations and deletion mutations affecting major and minor pilins. The extent of flocculation is increased in the hyperpiliated ΔpilT1 mutant, but active cycles of pilus extension and retraction are not required for flocculation. Deletion of PilA1, the major subunit of type IV pili, has no effect on flocculation; however, flocculation is lost in mutants lacking an operon coding for the minor pilins PilA9 to -11. Therefore, minor pilins appear crucial for flocculation. We show that flocculation is a tightly regulated process that is promoted by blue light perception by the cyanobacteriochrome Cph2. Floc formation also seems to be a highly cooperative process. A proportion of nonflocculating Δhfq cells can be incorporated into wild-type flocs, but the presence of a high proportion of Δhfq cells disrupts the large-scale architecture of the floc.IMPORTANCE Some bacteria form flocs, which are multicellular floating assemblages of many thousands of cells. Flocs have been relatively little studied compared to surface-adherent biofilms, but flocculation could play many physiological roles, be a crucial factor in marine carbon burial, and enable more efficient biotechnological cell harvesting. We studied floc formation and architecture in the model cyanobacterium Synechocystis sp. strain PCC 6803, using mutants to identify specific cell surface structures required for floc formation. We show that floc formation is regulated by blue and green light perceived by the photoreceptor Cph2. The flocs have a characteristic structure based on strands of linked cells aggregating into dense clusters. Cells within the dense clusters show signs of nutrient stress, pointing to a disadvantage of floc formation.

Using Nature's polyenes as templates: studies of synthetic xanthomonadin analogues and realising their potential as antioxidants.

Two truncated analogues of the polyenyl photoprotective xanthomonadin pigments have been synthesised utilising an iterative Heck-Mizoroki (HM)/iododeboronation cross coupling approach and investigated as models of the natural product photoprotective agents in bacteria. Despite the instability of these types of compounds, both analogues proved to be sufficiently stable to allow isolation, spectroscopic analysis and biological studies of their photoprotective behaviour which showed that despite their shorter polyene chain length, they retained the ability to protect bacteria from photochemical damage; i.e. incorporation of one compound into E. coli provided photoprotective activity against singlet oxygen analogous to the natural photoprotective mechanisms employed by Xanthomonas bacteria, answering key questions about what minimal functionality is required to impart photoprotection, potentially leading to new classes of photoprotective and antioxidants compounds.