IsProchlorothrix hollandica the best choice as a prokaryotic model for higher plant Chla/b photosynthesis?

We examine the issue of prochlorophyte origins and provide analyses which highlight the limitations of inferring evolutionary trees from anciently diverged sequences that have markedly different GC contents. Under these conditions we have found that current tree reconstruction methods strongly group together sequences with similar GC contents, whether or not the sequences share a common ancestor. We provide 3psbA termini sequence forProchloron didemni and find it does not have the 7 amino acid deletion that occurs in Chla/b chloroplasts andProchlorothrix hollandica. This is consistent with the recent findings of a Chlc like pigment in the light harvesting system in other prochlorophytes but apparently absent inP. hollandica. From these observations we suggest thatP. hollandica is the prochlorophyte most closely related to Chla/b containing chloroplasts and hence the most appropriate prokaryotic model for higher plant Chla/b photosynthesis.     

Domain structures of chlorophyllide<Emphasis Type="Italic"> a</Emphasis> oxygenase of green plants and<Emphasis Type="Italic"> Prochlorothrix hollandica</Emphasis> in relation to catalytic functions

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b–protein complexes in land plants.Abbreviations CAO Chlorophyllide a oxygenase - CP Chlorophyll protein - HPLC High-performance liquid chromatography - LHC Light-harvesting complex - PCR Polymerase chain reaction - PS Photosystem     

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.Abbreviations C carboxysome - CM cytoplasmic membrane - EF_s, EF_u exoplasmic fracture face of stacked and unstacked membrane area, respectively - ES exoplasmic surface - PF_s, PF_u plasmic fracture face of stacked and unstacked membrane area, respectively - PG peptidoglycan layer - TM thylakoid membrane Dedicated to Prof. Dr. D. Peters, Hamburg, on the occasion of his 75th birthday     

Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment-protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (> 200 μmol photons m⁻² s⁻¹) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment-protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem Q_B protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants. Xiaoping Jia and Jincheng Yuan have equal contribution.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them. Received: 20 July 1997 / Accepted: 7 October 1997     

云南不同高原湖泊噬藻体psbA基因多样性

【目的】噬藻体(cyanophage)广泛存在于自然水体生态系统中,通过侵染蓝藻进而调控蓝藻种群及群落结构,具有重要生态功能和生态地位,在控制蓝藻水华方面有巨大开发潜力。本研究旨在探究云南高原湖泊噬藻体psbA基因多样性,分析其系统进化地位,为深入了解高原湖泊生态功能、开发利用噬藻体资源奠定理论基础。【方法】以云南高原主要湖泊滇池、抚仙湖和星云湖等为研究对象,以psbA基因作为分子靶标,对湖泊水体中噬藻体遗传多样性进行研究。【结果】从不同湖泊中共获得100条环境噬藻体psbA基因序列,系统发育分析表明,湖泊的噬藻体psbA基因序列与中国东湖、中国东北稻田、日本稻田等淡水中的环境噬藻体psbA基因亲缘关系较近,与海洋环境噬藻体psbA基因亲缘关系较远;抚仙湖中的噬藻体psbA基因多样性高于滇池、星云湖和异龙湖中的噬藻体psbA基因多样性;云南高原湖泊中存在新的噬藻体类群;各湖泊秋冬季节噬藻体psbA基因遗传多样性差异不明显。【结论】云南主要高原湖泊噬藻体psbA基因遗传多样性高,与淡水环境噬藻体psbA基因亲缘关系较近,且存在独特的噬藻体类群。     

Zooplankton structure in the Loosdrecht lakes in relation to trophic status and recent restoration measures

A five-year zooplankton study (1982–86) on three shallow and highly eutrophic lakes in the Loosdrecht area (The Netherlands) did not reveal any significant changes following the considerable reduction in external P-loading (from about 1.0 g to 0.3 g P m^–2 year^–1) since mid-1984.The recent annual fluctuations in the rotifer and crustacean densities are within the range of those found before the restoration measure became operative. A decrease in the average size of the crustaceans and an absence of large-bodied forms reflects an increased fish predation rather than a change in the quality or quantity of their sestonic food ( < 150 µm) which continues to be dominated by filamentous cyanobacteria and Prochlorothrix hollandica, a prochlorophyte discovered in these lakes recently.     

A similar structure of the herbicide binding site in photosystem II of plants and cyanobacteria is demonstrated by site specific mutagenesis of the psbA gene

Many herbicides inhibit the photosynthetic electron transfer in photosystem II by binding to the polypeptide D1. A point mutation in the chloroplast gene psbA, which leads to a change of the amino acid residue 264 of D1 from serine to glycine, is responsible for atrazine resistance in higher plants. We have changed serine 264 to glycine in Synechococcus PCC7942 and compared its phenotype to a mutant with a serine to alanine shift in the same position. The results show that glycine at position 264 in D1 gives rise to a similar phenotype in cyanobacteria and in higher plants, indicating a similar structure of the binding site for herbicides and for the quinone Q_B in the two systems. A possible mode of binding of phenyl-urea herbicides to D1 is predicted from the difference in herbicidal cross-resistance between glycine and alanine substitutions of serine 264.Abbreviations DCPIP 2,6-dichlorophenolindophenol - I₅₀ concentration of herbicide giving 50% inhibition - K_b binding constant - kb kilobase - MES 2(N-morpholino)ethanesulfonic acid - PS II photosystem II     

Sequence analysis and phylogenetic reconstruction of the genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the chlorophyllb-containing prokaryoteProchlorothrix hollandica

Summary Prochlorophytes similar toProchloron sp. andProchlorothrix hollandica have been suggested as possible progenitors of the plastids of green algae and land plants because they are prokaryotic organisms that possess chlorophyllb (chlb). We have sequenced theProchlorothrix genes encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco),rbcL andrbcS, for comparison with those of other taxa to assess the phylogenetic relationship of this species. Length differences in the large subunit polypeptide among all sequences compared occur primarily at the amino terminus, where numerous short gaps are present, and at the carboxy terminus, where sequences ofAlcaligenes eutrophus and non-chlorophyllb algae are several amino acids longer. Some domains in the small subunit polypeptide are conserved among all sequences analyzed, yet in other domains the sequences of different phylogenetic groups exhibit specific structural characteristics. Phylogenetic analyses ofrbcL andrbcS using Wagner parsimony analysis of deduced amino acid sequences indicate thatProchlorothrix is more closely related to cyanobacteria than to the green plastid lineage. The molecular phylogenies suggest that plastids originated by at least three separate primary endosymbiotic events, i.e., once each leading to green algae and land plants, to red algae, and toCyanophora paradoxa. TheProchlorothrix rubisco genes show a strong GC bias, with 68% of the third codon positions being G or C. Factors that may affect the GC content of different genomes are discussed.     

Purification and characterization of a surface-associated carotenoid-binding complex from the photosynthetic prokaryote,Prochlorothrix hollandica

We have identified a water-soluble surface-associated complex from Prochlorothrix hollandica, composed of two polypeptides of 56 and 58 kilodaltons (kDa), zeaxanthin, and lipopolysaccharide. The complex was purified by preparative isoelectric focusing (pI=3.0). The outer membrane lipopolysaccharide co-purified with the complex. Immunocytochemisty employing a polyclonal antibody to the apoproteins exclusively labeled the cell surface. Both zeaxanthin and the protein accumulated under high light intensities, thus we propose that the complex may play a role in photoprotection.     

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola

Summary The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv Triton) has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the Triton psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position –9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The uncloneable nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.     

Analysis of heterologous regulatory and coding regions in algal chloroplasts

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5′ UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5′ UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.     

A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain

In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5- and 3-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe ^r) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe ^r phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (Q_A) to secondary quinone (Q_B) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (Q_A ^–).     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Characterization of the psbA Gene from Chloroplasts of Populus deltoides

A 2.9 kbp EcoRi fragment from chloroplast DNA of a tree species Populus deltoides, has been cloned. Nucleotide sequence analysis led to the identification of a 1062 bp open reading frame located at one end of the recombinant clone. This open reading frame has more than 94% nucleotide sequence homology with tobacco and cotton psbA genes. The deduced amino acid sequence from poplar psbA gene is highly homologous to tobacco and differs only by 2 amino acids located at C-terminus of the protein. An AT rich region, capable of forming a potential stem-loop structure was located down stream to the psbA gene. Our Southern hybridization data confirms the presence of IR region as well as the location of the psbA gene near one of the IR in P. deltoides.     

CYTOCHEMICAL STUDIES ON PROCHLOROPHYTES: LOCALIZATION OF DNA AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE-OXYGENASE1

We studied the distribution of the DNA-containing region and the ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCo) content of polyhedral bodies in three different prochlorophyte cell types in a search for broad evolutionary affinities of these chlorophyll b-containing prokaryotes. DNA was localized by DAPI staining and electron microscopy utilizing monoclonal anti-DNA antibody 2C-10 plus a secondary antibody labeled with colloidal gold. Antibodies against the large RuBisCo subunit from a higher plant raised in rabbits were used to localize RuBisCo in polyhedral bodies. We studied Prochloron Lewin cells from two different didemnid ascidian hosts (Lissoclinum patella and Didemnum molle) collected in Palau, West Caroline Islands, and cells of Prochlorothrix hollandica Burger-Wiersma, Stal, and Mur grown in laboratory culture. Cells of the blue-green alga Anabaena 7120 were studied for comparison. The DNA distribution was markedly different in the two Prochloron cell types. The thylakoids in cells from L. patella were concentrically arranged around a large central vacuole; the DNA-containing stromal areas appeared in thin sections as a concentric arcs between the thylakoid stacks. The central vacuole was lacking in cells from D. molle, and the thylakoid stacks and strands of DNA-containing stroma showed a more haphazard arrangement. In the filamentous Prochlorothrix the DNA-containing stroma was largely limited to a central nucleoid structure running the length of the cell. Although the DNA arrangements in Prochloron might be considered “chloroplast-like” since DNA-containing stroma is distributed, as in chloroplasts, in scattered sites among photosynthetic membranes, this is not so in Prochlorothrix, where there is an axial nucleoid, as in many filamentous cyanobacteria. Our anti-RuBisCo antibodies were selectively bound to the polyhedral bodies of all three cell types, indicating that Prochloron and Prochlorothrix, like many other autotrophic prokaryotes, possess typical carboxysomes.