Biochemical and molecular genetic approaches to studying protein export by cyanobacteria

We have shown that high molecular weight polypeptides are exported extracellularly by cyanobacteria. This is in addition to the well documented release of amino acids and peptides into the culture medium. Synechococcus R2 secretes two polypeptides of 14,400 molecular weight, and Nostoc sp. MAC a 43,000 molecular weight polypeptide. Their function is unclear, but we have some evidence that the 43,000 polypeptide may be involved in metal-binding. Outer membrane proteins (OMP), i.e. carotenoid-containing cell wall fractions, were isolated from several cyanobacteria and a prochlorophyte, and these showed that the extracellular polypeptides were not just cell wall components released into the culture medium. Functional analysis of Synechococcus R2 OMP showed that these proteins were heat-modifiable, a characteristic of porins, and that the pattern of OMP could be modulated by sucrose, magnesium limitation, phosphate- limitation and iron limitation. The effects of iron limitation on the OMP pattern could be simulated by the addition of EDDA (0.004%) to normal growth medium. Synechocystls sp. PCC6308 also Induced specific OMP under iron limitation. It is possible that Synechococcus R2 OMP are multifunctional. Antibodies raised against a Synechococcus R2 cell wall fraction cross-reacted with several other cyanobacterial OMP, and with an OMP fraction from Prochlorothrix hollandice. Using this antisera several positive plaques were obtained after screening a Synechococcus R2 Agtll library. Antibodies against an iron-regulated inner membrane protein, and a carotenoid-associated thylakoid protein cross-reacted with OMP from Synechococcus R2, suggesting a family of carotenoid- containing membrane proteins may exist. The lacZ gene, encoding the enzyme β-galactosidase, has been expressed in Synechococcus R2 on both a multicopy plasmid and when integrated into the cyanobacterial chromosome. The mechanism of integration relied on homologous recombination events between two pBR-plasmid derivatives. This required the construction of a universal recipient strain of Synechococcus R2. A gene encoding the extracellular enzyme pectate lyase from an Erwinia sp. was introduced into the chromosome of Synechococcus R2, but no detectable enzyme activity was observed. The transposon TnphoA was introduced into a cyanobacterial shuttle vector, but was incapable of transposition. Similarly, transposon Tn5, although stably maintained in the Synechococcus R2 chromosome after its introduction via homologous recombination, was not capable of transposition when introduced on a suicide vector. Even though transposition of Tn5 was observed in another Synechococcus sp. no mutant phenotypes were observed. NTG mutagenesis of Synechococcus R2 and Nostoc sp. MAC produced various pigment mutants, but mutants incapable of growth in chelator-deficient medium could not be obtained. This may reflect the selection procedure. The LacZ promoter probe has been constructed for use in Synechococcus R2 based on a multicopy plasmid capable of replicating in this organism. Differential expression of LacZ by Synechococcus R2 was observed under conditions of magnesium and iron limitation. Similar use of a kanamycin promoter probe suggests this construct will be useful for selecting strong promoter sequences via selection for high antibiotic resistance

Genetic diversity of eukaryotic ultraphytoplankton in the Gulf of Naples during an annual cycle

Eukaryotic ultraphytoplankton (<5 μm) are an important component of phytoplankton populations, Dot blot hybridisation analysis using class level 16S rRNA gene probes as well as clone libraries were used to investigate the diversity of these ultraphytoplankton during a 15 mo period (2003 to 2004) in the Gulf of Naples. Hybridisation data showed the presence of 3 main classes, Cryptophyceae, Chrysophyceae and Prymnesiophyceae, along with lower signals from the Pelagophyceae. Clone libraries also contained these 4 classes as well as sequences from the Dictyochophyceae, Bacillariophyceae and Prasinophyceae. However, the Prymnesiophyceae gave the dominant hybridisation signal and constituted the majority of each clone library. Their diversity, with a total of 190 sequences belonging to 114 operational taxonomic units (OTUs), probably allows them to dominate the ultraphytoplankton throughout the whole year under differing environmental conditions. Over 100 of these OTUs were unique to different libraries, suggesting a succession of different taxa during the year. The Cryptophyceae were present most of the year with 1 OTU, corresponding to a Plagioselmis prolonga strain from the Gulf of Naples, being the dominant taxon (28 % of sequences). A striking result was the high hybridisation signal from the Chrysophyceae, which showed a preference for the summer months. The Pelagophyceae were present between December and March. Most (80 %) of the sequences found in the clone libraries were not identical to available 16S rRNA gene sequences, indicating a high amount of hidden diversity for these algal classes. However, sequences from Prasinophyceae Clade II (Mamiellales) were not detected in the clone libraries

Unexpected evolutionary proximity of eukaryotic and cyanobacterial enzymes responsible for biosynthesis of retinoic acid and its oxidation

Biosynthesis of retinoic acid from retinaldehyde (retinal) is catalysed by an aldehyde dehydrogenase (ALDH) and its oxidation by cytochrome P450 enzymes (CYPs). Herein we show by phylogenetic analysis that the ALDHs and CYPs in the retinoic acid pathway in animals are much closer in evolutionary terms to cyanobacterial orthologs than would be expected from the standard models of evolution

Viruses inhibit CO2 fixation in the most abundant phototrophs on Earth

R.J.P. was the recipient of a NERC studentship and Warwick University IAS fellowship. This work was supported in part by NERC grant NE/J02273X/1 and Leverhulme Trust grant RPG-2014-354 to A.D.M., D.J.E., and D.J.S.Summary. Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus are the most numerous photosynthetic organisms on our planet [1, 2]. With a global population size of 3.6 × 1027 [3], they are responsible for approximately 10% of global primary production [3, 4]. Viruses that infect Prochlorococcus and Synechococcus (cyanophages) can be readily isolated from ocean waters [5–7] and frequently outnumber their cyanobacterial hosts [8]. Ultimately, cyanophage-induced lysis of infected cells results in the release of fixed carbon into the dissolved organic matter pool [9]. What is less well known is the functioning of photosynthesis during the relatively long latent periods of many cyanophages [10, 11]. Remarkably, the genomes of many cyanophage isolates contain genes involved in photosynthetic electron transport (PET) [12–18] as well as central carbon metabolism [14, 15, 19, 20], suggesting that cyanophages may play an active role in photosynthesis. However, cyanophage-encoded gene products are hypothesized to maintain or even supplement PET for energy generation while sacrificing wasteful CO2 fixation during infection [17, 18, 20]. Yet this paradigm has not been rigorously tested. Here, we measured the ability of viral-infected Synechococcus cells to fix CO2 as well as maintain PET. We compared two cyanophage isolates that share different complements of PET and central carbon metabolism genes. We demonstrate cyanophage-dependent inhibition of CO2 fixation early in the infection cycle. In contrast, PET is maintained throughout infection. Our data suggest a generalized strategy among marine cyanophages to redirect photosynthesis to support phage development, which has important implications for estimates of global primary production.Publisher PDFPeer reviewe

Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study

Background Marine Synechococcus owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrins I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine Synechococcus strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism. Results By carefully comparing the Synechococcus genomes, we have retrieved candidate genes potentially required for the synthesis of phycobiliproteins in each pigment type. This includes linker polypeptides, phycobilin lyases and a number of novel genes of uncharacterized function. Interestingly, strains belonging to a given pigment type have similar phycobilisome gene complements and organization, independent of the core genome phylogeny (as assessed using concatenated ribosomal proteins). While phylogenetic trees based on concatenated allophycocyanin protein sequences are congruent with the latter, those based on phycocyanin and phycoerythrin notably differ and match the Synechococcus pigment types. Conclusion We conclude that the phycobilisome core has likely evolved together with the core genome, while rods must have evolved independently, possibly by lateral transfer of phycobilisome rod genes or gene clusters between Synechococcus strains, either via viruses or by natural transformation, allowing rapid adaptation to a variety of light niches

Diel rhythmicity in amino acid uptake by Prochlorococcus

The marine cyanobacterium Prochlorococcus, the most abundant phototrophic organism on Earth, numerically dominates the phytoplankton in nitrogen (N)-depleted oceanic gyres. Alongside inorganic N sources such as nitrite and ammonium, natural populations of this genus also acquire organic N, specifically amino acids. Here, we investigated using isotopic tracer and flow cytometric cell sorting techniques whether amino acid uptake by Prochlorococcus is subject to a diel rhythmicity, and if so, whether this was linked to a specific cell cycle stage. We observed, in contrast to diurnally similar methionine uptake rates by Synechococcus cells, obvious diurnal rhythms in methionine uptake by Prochlorococcus cells in the tropical Atlantic. These rhythms were confirmed using reproducible cyclostat experiments with a light synchronised axenic Prochlorococcus (PCC9511 strain) culture and 35S-methionine and 3H-leucine tracers. Cells acquired the tracers at lower rates around dawn and higher rates around dusk despite >104 times higher concentration of ammonium in the medium, presumably because amino acids can be directly incorporated into protein. Leucine uptake rates by cells in the S+G2 cell cycle stage were consistently 2.2 times higher than those of cells at the G1 stage. Furthermore, S+G2 cells up-regulated amino acid uptake 3.5 times from dawn to dusk to boost protein synthesis prior to cell division. Because Prochlorococcus populations can account from 13% at midday, and up to 42% at dusk, of total microbial uptake of methionine and probably of other amino acids in N-depleted oceanic waters, this genus exerts diurnally variable, strong competitive pressure on other bacterioplankton populations

Draft genome sequence of Bacteriophage vB_Eco_swan01

Bacteriophage vB_Eco_swan01 was isolated from an ornamental pool using Escherichia coli MG1655 as the host. Bacteriophage vB_Eco_swan01 has limited similarity with other known phages at the nucleotide level and likely represents a new bacteriophage species within the Tunavirinae

PtrA is required for coordinate regulation of gene expression during phosphate stress in a marine Synechococcus

Previous microarray analyses have shown a key role for the two-component system PhoBR (SYNW0947, SYNW0948) in the regulation of P transport and metabolism in the marine cyanobacterium Synechococcus sp. WH8102. However, there is some evidence that another regulator, SYNW1019 (PtrA), probably under the control of PhoBR, is involved in the response to P depletion. PtrA is a member of the cAMP receptor protein transcriptional regulator family that shows homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the role of this regulator, we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-stress conditions. In response to P stress the ptrA mutant failed to upregulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is controlled by PtrA, including two phosphatases, a predicted phytase and a gene of unknown function psip1 (SYNW0165), all of which are highly upregulated during P limitation. Electrophoretic mobility shift assays indicate binding of overexpressed PtrA to promoter sequences upstream of the induced genes. This work suggests a two-tiered response to P depletion in this strain, the first being PhoB-dependent induction of high-affinity PO4 transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly influenced by PtrA, including those involved in cell-surface modification, metal uptake, photosynthesis, stress responses and other metabolic processes, which may indicate a wider role for PtrA in cellular regulation in marine picocyanobacteria

Accumulation of ambient phosphate into the periplasm of marine bacteria is proton motive force dependent

Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria

In situ associations between marine photosynthetic picoeukaryotes and potential parasites - a role for fungi?

Photosynthetic picoeukaryotes (PPEs) are important components of the marine picophytoplankton community playing a critical role in CO2 fixation but also as bacterivores, particularly in the oligotrophic gyres. Despite an increased interest in these organisms and an improved understanding of the genetic diversity of this group, we still know little of the environmental factors controlling the abundance of these organisms. Here, we investigated the quantitative importance of eukaryotic parasites in the free-living fraction as well as in associations with PPEs along a transect in the South Atlantic. Using tyramide signal amplification-fluorescence in situ hybridization (TSA-FISH), we provide quantitative evidence of the occurrence of free-living fungi in open ocean marine systems, while the Perkinsozoa and Syndiniales parasites were not abundant in these waters. Using flow cytometric cell sorting of different PPE populations followed by a dual-labelled TSA-FISH approach, we also demonstrate fungal associations, potentially parasitic, occurring with both pico-Prymnesiophyceae and pico-Chrysophyceae. These data highlight the necessity for further work investigating the specific role of marine fungi as parasites of phytoplankton to improve understanding of carbon flow in marine ecosystems