RESUMO
Carboxysomes are large self-assembled microcompartments that serve as the central machinery of a CO2-concentrating mechanism (CCM). Biogenesis of carboxysome requires the fine organization of thousands of individual proteins; however, the packaging pattern of internal RuBisCOs remains largely unknown. Here we purified the intact ß-carboxysomes from Synechococcus elongatus PCC 7942 and identified the protein components by mass spectrometry. Cryo-electron tomography combined with subtomogram averaging revealed the general organization pattern of internal RuBisCOs, in which the adjacent RuBisCOs are mainly arranged in three distinct manners: head-to-head, head-to-side, and side-by-side. The RuBisCOs in the outermost layer are regularly aligned along the shell, the majority of which directly interact with the shell. Moreover, statistical analysis enabled us to propose an ideal packaging model of RuBisCOs in the ß-carboxysome. These results provide new insights into the biogenesis of ß-carboxysomes and also advance our understanding of the efficient carbon fixation functionality of carboxysomes.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Ribulose-Bifosfato Carboxilase , Synechococcus , Synechococcus/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ribulose-Bifosfato Carboxilase/metabolismo , Ribulose-Bifosfato Carboxilase/química , Microscopia Crioeletrônica/métodos , Modelos MolecularesRESUMO
Carboxysomes are bacterial microcompartments that encapsulate the enzymes RuBisCO and carbonic anhydrase in a proteinaceous shell to enhance the efficiency of photosynthetic carbon fixation. The self-assembly principles of the intact carboxysome remain elusive. Here we purified α-carboxysomes from Prochlorococcus and examined their intact structures using single-particle cryo-electron microscopy to solve the basic principles of their shell construction and internal RuBisCO organization. The 4.2 Å icosahedral-like shell structure reveals 24 CsoS1 hexamers on each facet and one CsoS4A pentamer at each vertex. RuBisCOs are organized into three concentric layers within the shell, consisting of 72, 32 and up to 4 RuBisCOs at the outer, middle and inner layers, respectively. We uniquely show how full-length and shorter forms of the scaffolding protein CsoS2 bind to the inner surface of the shell via repetitive motifs in the middle and C-terminal regions. Combined with previous reports, we propose a concomitant 'outside-in' assembly principle of α-carboxysomes: the inner surface of the self-assembled shell is reinforced by the middle and C-terminal motifs of the scaffolding protein, while the free N-terminal motifs cluster to recruit RuBisCO in concentric, three-layered spherical arrangements. These new insights into the coordinated assembly of α-carboxysomes may guide the rational design and repurposing of carboxysome structures for improving plant photosynthetic efficiency.
RESUMO
The unicellular picocyanobacterium Prochlorococcus is the most abundant photoautotroph and contributes substantially to global CO2 fixation. In the vast euphotic zones of the open ocean, Prochlorococcus converts CO2 into organic compounds and supports diverse organisms, forming an intricate network of interactions that regulate the magnitude of carbon cycling and storage in the ocean. An understanding of the biological interactions with Prochlorococcus is critical for accurately estimating the contributions of Prochlorococcus and interacting organisms to the marine carbon cycle. This review synthesizes the primary production contributed by Prochlorococcus in the global ocean. We outline recent progress on the interactions of Prochlorococcus with heterotrophic bacteria, phages, and grazers that multifacetedly determine Prochlorococcus carbon production and fate. We discuss that climate change might affect the biological interactions with Prochlorococcus and thus the marine carbon cycle.
Assuntos
Prochlorococcus , Prochlorococcus/fisiologia , Dióxido de Carbono , Bactérias , Ciclo do Carbono , Carbono , Água do Mar/microbiologiaRESUMO
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.