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Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibers that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Three further gene products with similarity to PilX-like minor pilins have a function in flocculation of Synechocystis. The results of our study indicate that different minor pilins facilitate distinct pilus functions. Further, our microarray analysis demonstrated that the transcription levels of the minor pilin genes change in response to surface contact. A total of 122 genes were determined to have altered transcription between planktonic and surface growth, including several plasmid genes which are involved exopolysaccharide synthesis and the formation of bloom-like aggregates.

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibres that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. The transcription of the minor pilin genes pilA5, pilA6 and pilA9-pilA11 is inversely regulated in response to different conditions. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Microarray analysis demonstrated that the transcription levels of known and newly predicted minor pilin genes change in response to surface contact. A total of 120 genes were determined to have altered transcription between planktonic and surface growth. Among these genes, 13 are located on the pSYSM plasmid. The results of our study indicate that different minor pilins facilitate distinct pilus functions.

Crocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean., Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2. Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2. The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m−2 s−1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m−2 s−1. This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface. IMPORTANCE Crocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

Algae of the genus Nannochloropsis are attractive organisms for use in biotechnology due to their high lipid content. Genetic manipulation of marine Nannochloropsis species has already been reported; however, tools have not yet been developed to transform Nannochloropsis limnetica, the only known freshwater species of this genus. To establish N. limnetica as a model laboratory strain, we first tested the effects of 11 different antibiotics on growth of N. limnetica and the marine species N. oceanica and N. gaditana. These three microalgae responded very differently to antibiotic treatments, both in liquid cultures and on agar plates. In general, N. limnetica exhibited a much higher sensitivity to antibiotics than the marine strains, thus offering the potential for a large set of antibiotic resistance genes that may be applicable as artificial selection markers after transformation. We also developed a simple protocol using lysozyme to obtain high yields of viable N. limnetica protoplasts, as confirmed by flow cytometry and electron microscopy.

A characteristic feature of the active Photosystem II (PSII) complex is a red-shifted low temperature fluorescence emission at about 693 nm. The origin of this emission has been attributed to a monomeric 'red' chlorophyll molecule located in the CP47 subunit. However, the identity and function of this chlorophyll remain uncertain. In our previous work, we could not detect the red PSII emission in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking PsbH, a small transmembrane subunit bound to CP47. However, it has not been clear whether the PsbH is structurally essential for the red emission or the observed effect of mutation has been indirectly caused by compromised PSII stability and function. In the present work we performed a detailed spectroscopic characterization of PSII in cells of a mutant lacking PsbH and Photosystem I and we also characterized PSII core complexes isolated from this mutant. In addition, we purified and characterized the CP47 assembly modules containing and lacking PsbH. The results clearly confirm an essential role of PsbH in the origin of the PSII red emission and also demonstrate that PsbH stabilizes the binding of one β-carotene molecule in PSII. Crystal structures of the cyanobacterial PSII show that PsbH directly interacts with a single monomeric chlorophyll ligated by the histidine 114 residue of CP47 and we conclude that this peripheral chlorophyll hydrogen-bonded to PsbH is responsible for the red fluorescence state of CP47. Given the proximity of β-carotene this state could participate in the dissipation of excessive light energy.

Summary The molecular and physiological mechanisms involved in the transition of microbial cells from a resting state to the active vegetative state are critically relevant for solving problems in fields ranging from microbial ecology to infection microbiology. Cyanobacteria that cannot fix nitrogen are able to survive prolonged periods of nitrogen starvation as chlorotic cells in a dormant state. When provided with a usable nitrogen source, these cells re-green within 48 hr and return to vegetative growth. Here we investigated the resuscitation of chlorotic Synechocystis sp. PCC 6803 cells at the physiological and molecular levels with the aim of understanding the awakening process of a dormant bacterium. Almost immediately upon nitrate addition, the cells initiated a highly organized resuscitation program. In the first phase, they suppressed any residual photosynthetic activity and activated respiration to gain energy from glycogen catabolism. Concomitantly, they restored the entire translational apparatus, ATP synthesis, and nitrate assimilation. After only 12–16 hr, the cells re-activated the synthesis of the photosynthetic apparatus and prepared for metabolic re-wiring toward photosynthesis. When the cells reached full photosynthetic capacity after ∼48 hr, they resumed cell division and entered the vegetative cell cycle. An analysis of the transcriptional dynamics during the resuscitation process revealed a perfect match to the observed physiological processes, and it suggested that non-coding RNAs play a major regulatory role during the lifestyle switch in awakening cells. This genetically encoded program ensures rapid colonization of habitats in which nitrogen starvation imposes a recurring growth limitation.

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use (35)S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b 6 f and the HliD- Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed.

Summary Type IV pilins are bacterial proteins that are small in size but have a broad range of functions, including motility, transformation competence and secretion. Although pilins vary in sequence, they possess a characteristic signal peptide that has to be removed by the prepilin peptidase PilD during pilin maturation. We generated a pilD (slr1120) null mutant of the cyanobacterium Synechocystis 6803 that accumulates an unprocessed form of the major pilin PilA1 (pPilA1) and its non-glycosylated derivative (NpPilA1). Notably, the pilD strain had aberrant membrane ultrastructure and did not grow photoautotrophically because the synthesis of Photosystem II subunits was abolished. However, other membrane components such as Photosystem I and ATP synthase were synthesized at levels comparable to the control strain. Proliferation of the pilD strain was rescued by elimination of the pilA1 gene, demonstrating that PilA1 prepilin inhibited the synthesis of Photosystem II. Furthermore, NpPilA1 co-immunoprecipitated with the SecY translocase and the YidC insertase, and both of these essential translocon components were degraded in the mutant. We propose that unprocessed prepilins inactivate an identical pool of translocons that function in the synthesis of both pilins and the core subunits of Photosystem II.

Type IV pilins are bacterial proteins that are small in size but have a broad range of functions, including motility, transformation competence and secretion. Although pilins vary in sequence, they possess a characteristic signal peptide that has to be removed by the prepilin peptidase PilD during pilin maturation. We generated a pilD (slr1120) null mutant of the cyanobacterium Synechocystis 6803 that accumulates an unprocessed form of the major pilin PilA1 (pPilA1) and its non-glycosylated derivative (NpPilA1). Notably, the pilD strain had aberrant membrane ultrastructure and did not grow photoautotrophically because the synthesis of Photosystem II subunits was abolished. However, other membrane components such as Photosystem I and ATP synthase were synthesized at levels comparable to the control strain. Proliferation of the pilD strain was rescued by elimination of the pilA1 gene, demonstrating that PilA1 prepilin inhibited the synthesis of Photosystem II. Furthermore, NpPilA1 co-immunoprecipitated with the SecY translocase and the YidC insertase, and both of these essential translocon components were degraded in the mutant. We propose that unprocessed prepilins inactivate an identical pool of translocons that function in the synthesis of both pilins and the core subunits of Photosystem II.

Cyanobacteria acclimate to high-light conditions by adjusting photosystem stoichiometry through a decrease of photosystem I (PSI) abundance in thylakoid membranes. As PSI complexes bind the majority of chlorophyll (Chl) in cyanobacterial cells, it is accepted that the mechanism controlling PSI level/synthesis is tightly associated with the Chl biosynthetic pathway. However, how Chl is distributed to photosystems under different light conditions remains unknown. Using radioactive labeling by (35)S and by (14)C combined with native/two-dimensional electrophoresis, we assessed the synthesis and accumulation of photosynthetic complexes in parallel with the synthesis of Chl in Synechocystis sp. PCC 6803 cells acclimated to different light intensities. Although cells acclimated to higher irradiances (150 and 300 μE m(-2)s(-1)) exhibited markedly reduced PSI content when compared with cells grown at lower irradiances (10 and 40 μE m(-2) s(-1)), they grew much faster and synthesized significantly more Chl, as well as both photosystems. Interestingly, even under high irradiance, almost all labeled de novo Chl was localized in the trimeric PSI, whereas only a weak Chl labeling in photosystem II (PSII) was accompanied by the intensive (35)S protein labeling, which was much stronger than in PSI. These results suggest that PSII subunits are mostly synthesized using recycled Chl molecules previously released during PSII repair-driven protein degradation. In contrast, most of the fresh Chl is utilized for synthesis of PSI complexes likely to maintain a constant level of PSI during cell proliferation.