F. Gerald Plumley

Light-Harvesting Chlorophyll a/b Complexes: Interdependent Pigment Synthesis and Protein Assembly.

The biogenetic interdependence of light-harvesting chlorophyll (Chl) a/b proteins (LHCPs) and antenna pigments has been analyzed for two nuclear mutants of Chlamydomonas that have low levels of Chl b, neoxanthin, and loroxanthin. In mutant PA2.1, the apoprotein precursors (pLHCP II) of the major light-harvesting complex LHC II were synthesized at approximately wild-type rates, processed to their mature size, and rapidly degraded. Because the bulk of labile LHCP II in PA2.1 was soluble, a thylakoid integration factor apparently is defective in this strain. Chl a, Chl b, neoxanthin, and loroxanthin synthesis and accumulation were coordinately reduced in PA2.1, indicating that LHCP II play important regulatory or substrate roles in de novo synthesis of these pigments. Mutant GE2.27 is impaired principally in Chl b synthesis but nonetheless accumulated wild-type levels of all LHCPs. Topology studies of the GE2.27 LHCP II demonstrated that their insertion into thylakoids was incomplete even though they were not structurally altered. Thus, Chl b formation mediates conformational changes of LHCP II after thylakoid integration is initiated. GE2.27 also exhibited very low rates of neoxanthin synthesis and was unable to accumulate loroxanthin. Revertant GE2.27 strains with varying capacities for Chl b formation provided additional evidence that neoxanthin synthesis and accumulation are coupled with the final steps of LHCP II integration into thylakoids. We propose that biogenesis of LHC includes interdependent pigment synthesis/assembly events that occur during LHCP integration into the thylakoid membrane and that defects in these events account for the pleiotropic characteristics of many Chl b-deficient mutants.

Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria

Background Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. Methodology/Principal Findings We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxingenes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX−) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. Conclusions/Significance Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX− strains among Anabaena circinalis strains.

Evolution of Saxitoxin Synthesis in Cyanobacteria and Dinoflagellates

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

GTX4 imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light‐harvesting antenna of photosystem I or II, or a chlorophyll‐protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically‐related proteins correlates with the quantity of light‐harvesting chlorophyll‐protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c‐binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl‐binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.

Oxygen-evolving diatom thylakoid membranes.

Two protocols were developed that yielded purified oxygen-evolving thylakoid membranes from the diatom Cylindrotheca fusiformis. One protocol employed sonication, while the second involved French press lysis of protoplasts formed by brief culture of cells in a cation-depleted medium. Regardless of the method of cell breakage, some damage to electron transport components occurred. For preservation of both light-dependent electron transport activity and in vivo fluorescence properties, 2 M sorbitol proved to be more effective than 1 M sorbitol, regardless of the method used for cell lysis. Thylakoids purified in 2 M sorbitol using the protoplast/French press method showed the best preservation of in vivo fluorescence emission signals and Photosystem II activity with ferricyanide was completely inhibited by DCMU. Thylakoids purified in 2 M sorbitol using sonication had higher rates of Photosystem II activity with ferricyanide, but this activity was less sensitive to DCMU. Whole-chain electron transport activity was low in all preparations. This is the first report of O2 evolution and of long-wavelength fluorescence in purified thylakoids of any chromophytic alga.

Growth and pigmentation of juvenile Porphyra torta (Rhodophyta) gametophytes in response to nitrate, salinity and inorganic carbon

The leafy gametophytic phase of Porphyra torta Krishnamurthy(Rhodophyta), a candidate species for mariculture in Alaska, grows only inwinter and early spring and is restricted to the outer coast of southeastAlaska. To help determine specific environmental factors limiting theseasonal and geographic distribution of this species, culture experimentswere conducted using environmentally realistic levels of three physicalfactors. Growth and phycoerythrin concentration in juvenile gametophyteswere compared under combinations of nitrate, salinity, and inorganiccarbon representing the maximum and minimum levels of each factor in themarine environment. Recovery experiments were also conducted todetermine whether blades affected by low nutrient or salinity levels couldregain normal growth rates and pigment levels. To make statistically validcomparisons of growth rates among treatment groups, where repeatedmeasures were used, a two-stage analysis was tested and found to beappropriate. Low nitrate had a significant, negative effect on growth andphycoerythrin concentration. Salinity had a weak, negative effect on bladegrowth, while inorganic carbon had no observed effect on blade growth,and neither had a significant effect on phycoerythrin concentration. Bladesaffected by low nitrate were able to regain normal growth rates and higherthan normal pigment levels when nitrate was increased, after up to 6 weeksof exposure. The growth rate, modeled from the data, increased with timeinitially, dependent on nitrate level.

Tn5 MUTAGENESIS OF PSEUDOMONAS STUTZERI SF/PS, A BACTERIUM ASSOCIATED WITH ALEXANDRIUM LUSITANICUM (DINOPHYCEAE) AND PARALYTIC SHELLFISH POISONING

It is becoming increasingly clear that bacteria can play an important role in the toxin and population dynamics of harmful algal bloom (HAB) events. In this paper, we document protocols and strategies that can be used to identify bacterial genes involved in either the production of toxic compounds and/or the establishment and maintenance of relationships between bacteria and algae. The protocols we tested involved transposon mutagenesis and complementation with broad‐host‐range plasmids. We tested six bacterial strains thought to be involved, either directly or indirectly, in the production of toxins associated with paralytic shellfish poisoning (PSP). Five strains were resistant to transformation under the growth conditions used. However, a single strain, Pseudomonas stutzeri SF/PS, was readily transformed when grown under appropriate conditions. This bacterium has been shown to accumulate PSP toxins and to increase toxin production when added to axenic cultures of a toxic dinoflagellate, Alexandrium lusitanicum. We conclude that a transposon mutagenesis strategy can be used to identify genes involved in HAB events.