Gerard Guglielmi

The structure of cyanobacterial phycobilisomes: a model

Phycobilisomes, supramolecular complexes of water-soluble accessory pigments, serve as the major light-harvesting antennae in cyanobacteria and red algae. Regular arrays of these organelles are found on the surface of the thylakoid membranes of these organisms. In the present study, the hemi-discoidal phycobilisomes of several species of cyanobacteria were examined in thin sections of cells and by negative staining after isolation and fixation. Their fundamental structures were found to be the same. Isolated phycobilisomes possessed a triangular core assembled from three stacks of disc-shaped subunits. Each stack contained two discs which were ∼12 nm in diameter and ∼6–7 nm thick. Each of these discs was probably subdivided into halves ∼3–3.5 nm thick. Radiating from each of two sides of the triangular core were three rods ∼12 nm in diameter. Each rod consisted of stacks of 2 to 6 disc-shaped subunits ∼6 nm thick. These discs were subdivided into halves ∼3 nm thick.The average number of discs of ∼6 nm thickness forming the peripheral rods varied among the strains studied. For certain chromatically adapting strains, the average rod length was dependent upon the wavelength of light to which cells were exposed during growth. Analyses of phycobilisomes by spectroscopic techniques, polyacrylamide gel electrophoresis, and electron microscopy were compared. These analyses suggested that the triangular core was composed of allophycocyanin and that the peripheral rods contained phycocyanin and phycoerythrin (when present). A detailed model of the hemi-discoidal phycobilisome is proposed. This model can account for many aspects of phycobiliprotein assembly and energy transfer.

The structure of gloeobacter violaceus and its phycobilisomes

The fine structure of the atypical cyanobacterium Gloeobacter violaceus has been studied on frozen-etched replicas and compared to that of a typical unicellular strain: Synechocystis 6701. The complementary fracture faces of G. violaceus cytoplasmic membrane contain particles less numerous and more heterogenous in size than either the cytoplasmic membrane or the thylakoid membranes of Synechocystis. The most frequently observed particles of the exoplasmic fracture (EF) face of the G. violaceus cytoplasmic membrane are 11 nm in diameter and occasionally form short alignments. This particle class is similar in appearance to the numerous, aligned EF particles of Synechocystis thylakoid membranes. In replicas of cross-fractured G. violaceus, a layer 50–70 nm thick, composed of rod-like elements, underlies the inner surface of the cytoplasmic membrane. The rods, 12–14 nm in diameter, are oriented perpendicularly to the cytoplasmic membrane and show a 6 nm repeat along their length.Isolated phycobilisomes of G. violaceus appear, after fixation and negative staining, as bundles of 6 parallel rodshaped elements connected to an ill-defined basal structure. The bundles are 40–45 nm wide and 75–90 nm long. The rods are 10–12 nm in width; their length varies between 50 and 70 nm. These rods are morphologically similar to those observed at the periphery of hemidiscoidal phycobilisomes of other cyanobacteria, with a strong repeat at 6 nm intervals and a weaker one at 3 nm intervals along their length.The calculated molar ratio of phycobiliproteins in isolated G. violaceus phycobilisomes corresponds to 1:3.9:2.9 for allophycocyanin, phycocyanin and phycoerythrin respectively. When excited at 500 nm, isolated phycobilisomes exhibit a major fluorescence emission band centered at 663 nm.

A developmentally regulated gvpABC operon is involved in the formation of gas vesicles in the cyanobacterium Calothrix 7601.

In the filamentous cyanobacterium Calothrix PCC7601, gas-vesicle (GV) formation is restricted to specialized filaments, called hormogonia. The differentiation of these cells is controlled by environmental factors, such as light intensity and/or wavelength. The structural gene (gvpA) encoding a GV protein in this cyanobacterium has been previously cloned and sequenced. Two other genes, gvpB and gvpC have been found in the sequence downstream from gvpA. The gvpB gene corresponds to a second copy of gvpA, encoding an identical protein. Unlike the GV protein, the product of the gvpC gene is predominantly hydrophilic, as deduced from nucleotide sequence. Interestingly, the internal part of the gvpC gene is composed of four contiguous repeats, each containing 99 bp, forming highly homologous repeats in the deduced amino acid sequence. Another kind of periodicity has been detected inside the 99-bp repeats, suggesting that the gvpC gene might have evolved by amplification of a 33-bp-long primordial building block. The function of this gene remains to be elucidated. Finally, we have shown that the three genes, gvpA, gvpB, and gvpC, are organized in an operon that is exclusively expressed during GV formation in hormogonia.

Photoregulation of gene expression in the filamentous cyanobacterium Calothrix sp. PCC 7601: light-harvesting complexes and cell differentiation

Light plays a major role in many physiological processes in cyanobacteria. In Calothrix sp. PCC 7601, these include the biosynthesis of the components of the light-harvesting antenna (phycobilisomes) and the differentiation of the vegetative trichomes into hormogonia (short chains of smaller cells). In order to study the molecular basis for the photoregulation of gene expression, physiological studies have been coupled with the characterization of genes involved either in the formation of phycobilisomes or in the synthesis of gas vesicles, which are only present at the hormogonial stage.In each system, a number of genes have been isolated and sequenced. This demonstrated the existence of multigene families, as well as of gene products which have not yet been identified biochemically. Further studies have also established the occurrence of both transcriptional and post-transcriptional regulation. The transcription of genes encoding components of the phycobilisome rods is light-wavelength dependent, while translation of the phycocyanin genes may require the synthesis of another gene product irrespective of the light regime. In this report, we propose two hypothetical models which might be part of the complex regulatory mechanisms involved in the formation of functional phycobilisomes. On the other hand, transcription of genes involved in the gas vesicles formation (gvp genes) is turned on during hormogonia differentiation, while that of phycobiliprotein genes is simultaneously turned off. In addition, and antisense RNA which might modulate the translation of the gvp mRNAs is synthezised.

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis.

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin α and β subunits; allophycocyanin α and β subunits; α subunit of allophycocyanin B; the allophycocyanin β-subunit-like polypeptide of Mr 18 000; the linker phycobiliprotein of Mr 99 000; and non-chromophore-carrying linker polypeptides of Mr 33 000, 29 000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin α and β subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2–7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.

Structure and mutation of a gene encoding a Mr 33000 phycocyanin-associated linker polypeptide

The gene encoding a phycocyanin-associated linker polypeptide of Mr 33000 from the cyanobacterium Synechococcus sp. PCC 7002 was found to be located adjacent and 3′ to the genes encoding the α and β subunits of phycocyanin. The identity of this gene, designated cpcC, was proven by matching the amino-terminal sequence of the authentic polypeptide with that predicted by the nucleotide sequence. A cpcC mutant strain of this cyanobacterium was constructed. The effect of the mutation was to prevent assembly of half the total phycocyanin into phycobilisomes. By electron microscopy, phycobilisomes from this mutant were shown to contain rod substructures composed of a single disc of hexameric phycocyanin, as opposed to two discs in the wild type. It was concluded that the Mr 33000 linker polypeptide is required for attachment of the core-distal phycocyanin hexamer to the core-proximal one. Using absorption spectra of the wild type, CpcC−, and phycocyanin-less phycobilisomes, the in situ absorbances expected for specific phycocyanin-linker complexes were calculated. These data confirm earlier findings on isolated complexes regarding the influence of linkers on the spectroscopic properties of phycocyanin.

Phycobilisomes of the Cyanobacterium Synechococcus SP. PCC 7002: Structure, Function, Assembly, and Expression

The cyanobacterial photosynthetic apparatus is remarkably similar in structure and function to that found in the chloroplasts of eucaryotic algae and higher plants (Bryant, 1987). Four major multiprotein complexes of the thylakoids—the Photosystem II complex = the water-plastoquinone photo-oxidoreductase; the cytochrome b6/f complex= the plastoquinol-plastocyanin (cytochrome c553) oxidoreductase; the Photosystem I complex = plastocyanin (cytochrome c553)-ferredoxin (flavodoxin) photo-oxidoreductase; and the ATP synthase—have been shown to be rather similar in all oxygenic procaryotes and eucaryotes studied. The predominant differences among the photosynthetic apparatuses in the various algae and higher plants derives from the considerable diversity that exists in the light-harvesting antennae systems among these organisms. In eucaryotic algae and higher plants, the light-harvesting complexes for Photosystem I and Photosystem II are a diverse collection of carteno-chlorophyll protein complexes that in general are integral membrane components (Owens, 1988; Thornber et al., 1988). Such antenna systems are also found in certain procaryotes such as Prochloron sp. and Prochlorothrix hollandica (Bullerjahn et al., 1987). However, in the cyanobacteria, in the chloroplasts of the eucaryotic red algae, and in the cyanelles of certain phylogenetically ambiguous eucaryotes such as Cyanophora paradoxa, the light-harvesting antenna complexes for Photosystem II are large, multiprotein complexes composed of water-soluble proteins, the phycobilisomes, which are attached to the thylakoid surface in close proximity to the Photosystem II reaction centers (Bryant, 1987).

The Cyanobacterial Photosynthetic Apparatus: A Structural and Functional Analysis Employing Molecular Genetics

A major conclusion from recent detailed analyses of the cyanobacterial and higher plant photosynthetic apparatuses is that these two systems are remarkably conserved both structurally and functionally (1). The cyanobacterial photosynthetic apparatus, like that of eucaryotic algae and higher plants, is largely composed of five supramolecular, multiprotein assemblies: 1. the Photosystem II/P680/Oxygen Evolution Complex (water-plastoquinone photo-oxidoreductase); 2. the plastoquinol-plastocyanin oxidoreductase (cytochrome b 6/f complex); 3. the Photosystem I/P700 complex (plastocyanin-ferredoxin photo-oxidoreductase); the ATP synthase complex (Coupling Factor); and 5. light-harvesting antennae complex(es). For detailed reviews of the cyanobacterial and higher plant photosynthetic apparatuses, the reader should consult references 1 and 2. In eyanobac-teria the light-harvesting antenna complex is a large, thylakoid-extrinsic protein complex called the phycobilisome (1, 3, 4). In higher plants these antenna complexes are comprised of the nuclear-encoded chlorophyll a/b proteins which are a family of intrinsic thylakoid proteins which are associated with both Photosystem I and Photosystem II (2, 5).