Complete amino acid sequence of cyanobacterial gas-vesicle protein indicates a 70-residue molecule that corresponds in size to the crystallographic unit cell.

Gas vesicles of cyanobacteria are formed by a protein called 'gas-vesicle protein' (GVP). The complete amino acid sequence has been determined of GVP from Anabaena flos-aquae. It is 70 residues long and has an Mr of 7388. This corresponds to the size of the repeating unit cell demonstrated by X-ray crystallography of intact gas vesicles. Details of the sequence are related to the secondary beta-sheet structure of the protein and its contrasting hydrophilic and hydrophobic surfaces. Extensive amino acid sequences have also been determined for GVPs from two other cyanobacteria, species of Calothrix and Microcystis; they are highly homologous with that of Anabaena GVP. Electrophoretic analysis indicates that GVPs of different cyanobacteria form a variety of stable oligomers.     

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.     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

Occurrence and distribution of gas vesicle genes among cyanobacteria.

Gas vesicles (GV) are specialized cell inclusions providing many aquatic procaryotes with buoyancy. In the cyanobacterium Calothrix sp. strain PCC 7601, at least four genes are involved in GV formation. One of those, gvpA1, encodes the major structural GV protein (70 amino acids) and belongs to a multigene family (gvpA1, gvpA2, gvpD). The fourth gene, gvpC, encodes a 162-amino-acid protein, the function of which is still unclear. We used the Calothrix gvpA1 and gvpC genes as probes to perform Southern hybridization experiments with DNA extracted from various cyanobacterial strains. The gvpA gene was found in all the strains that synthesize GV, indicating that its product is an obligatory component of GV. Furthermore, it was found to occur as multiple copies in most of the strains tested. The gvpC gene was only detected in some strains able to synthesize a large amount of GV within a short period. This suggests that the gvpC gene product is a dispensable protein for GV formation and is involved in the efficiency of the assembly process. Based on the occurrence of the gvp genes and on DNA-DNA hybridization patterns, genus assignments are discussed.     

Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria.

The genes encoding the RNA subunit of ribonuclease P from the unicellular cyanobacterium Synechocystis sp. PCC 6803, and from the heterocyst-forming strains Anabaena sp. PCC 7120 and Calothrix sp. PCC 7601 were cloned using the homologous gene from Anacystis nidulans (Synechococcus sp. PCC 6301) as a probe. The genes and the flanking regions were sequenced. The genes from Anabaena and Calothrix are flanked at their 3'-ends by short tandemly repeated repetitive (STRR) sequences. In addition, two other sets of STRR sequences were detected within the transcribed regions of the Anabaena and Calothrix genes, increasing the length of a variable secondary structure element present in many RNA subunits of ribonuclease P from eubacteria. The ends of the mature RNAs were determined by primer extension and RNase protection. The predicted secondary structure of the three RNAs studied is similar to that of Anacystis and although some idiosyncrasies are observed, fits well with the eubacterial consensus.     

水华鱼腥藻类菌胞素氨基酸的分子鉴定和化学检测

类菌胞素氨基酸 (MAAs) 是一类具有吸收紫外线能力的物质, 蓝藻MAAs的生物合成及其分子机制的揭示为MAAs基因的快速检测提供了可能。研究采用分子生物学方法扩增了一株分离自太湖的水华鱼腥藻(Dolichospermum flos-aquae CHAB1629)编码脱氢醌合成酶(DHQS)基因的部分片段, 系统树分析发现其与报道的Anabaena sp. 90核酸序列相似度达99%, 而与Anabaena variabilis ATCC 29413仅为53.6%; 同时运用HPLC检测发现, 该株水华鱼腥藻MAAs的类型为shinorine。研究结果可为后续浮游类鱼腥藻MAAs的分子鉴定及野外适应性研究提供依据。       

Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac-region boundary and suggests a regulatory role for the gvpD gene or its product

A series of deletions introduced into the gvp gene cluster of Haloferax mediterranei, comprising 14 genes involved in gas vesicle synthesis (mc-vac-region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc-vac-region produced gas vesicles (Vac+), various deletions in the region 5' to gvpA (encompassing gvpD-gvpM) or 3' to gvpA (containing gvpC, gvpN and gvpO) revealed Vac- transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.     

Biosynthesis of the tropane-related cyanobacterial toxin anatoxin-a: role of ornithine decarboxylase.

A study was made of the biosynthesis by Anabaena flos-aquae of the tropane-related alkaloid anatoxin-a. Evidence is presented that the toxin arises from ornithine via putrescine (1,4-diaminobutane) and that ornithine decarboxylase (EC 4.1.1.17) is involved. An ornithine decarboxylase preparation, with optimal activity at pH 8, was obtained from Anabaena flos-aquae and partially purified by gel-filtration chromatography on DEAE-cellulose. One major and one minor peak of enzymic activity were obtained with Km values of 1.25 and 2.5 mM, respectively. Plasmid DNA (10 Kb; Mr 6.5 x 10(6] was detected in the toxic strain of Anabaena flos-aquae but not in a non-toxic strain. DNA from the toxin-producing strain of Anabaena flos-aquae transforms the non-toxic into a toxic strain.     

Molecular Characterization of a Fungicidal Endoglucanase from the Cyanobacterium Calothrix elenkinii

A gene responsible for fungicidal activity was identified in the cyanobacterial strain Calothrix elenkinii RPC1, which had shown promise as a biocontrol agent. Functional screening of the genomic library revealed fungicidal (against Pythium aphanidermatum) and endoglucanase activities in two clones. Sequencing revealed an open reading frame of 1,044 bp, encoding 348 amino acid residues with a predicted molecular weight of 38 kDa. Analysis of the deduced amino acid sequence of the putative gene (cael1) showed 99% similarity with the β-1,4-endoglucanase from Anabaena laxa RPAN8 and 97% with the glucanase belonging to the peptidase M20 family of Anabaena variabilis and Nostoc sp. PCC7120, respectively. The putative promoters, ribosomal binding sites and a signal peptide of 22 amino acid residues were identified, revealing the secretory nature of the protein. The phylogenetic tree indicated a close relationship of the gene with Bacillus sp. This study is the first to report on the characterization of an endoglucanase in Calothrix sp.     

General distribution of the nitrogen control gene ntcA in cyanobacteria.

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.     

Isolation and verification of anatoxin-a producing clones of Anabaena flos-aquae (Lyngb.) de Breb. from a eutrophic lake

Abstract We present a technique to isolate and confirm anatoxin-a producing clones (single trichome-isolates) of Anabaena flos-aquae (Lyngb.) de Breb. from blooms of this cyanobacterium. A single trichome is isolated from a field sample and grown in ASM medium. Single trichomes are then isolated from this culture and grown in ASM medium to produce single clone cultures. Mouse bioassay, and thin-layer chromatography (TLC) using purified anatoxin-a as reference is then used to confirm the anatoxin-a producing clones. Using this methodology, Anabaena flos-aquae samples collected during July 1991 from Hebgen Lake, Montana, were found to contain only 8.7% anatoxin-a producing clones. This minor proportion of anatoxin-a producing clones apparently accounts for the anatoxin-a produced by the entire population of A. flos-aquae . Our technique is simple and reproducible. A selected clone of A. flos-aquae that produces anatoxin-a and one that does not produce anatoxin-a were deposited in the UTEX culture collection, University of Texas at Austin.     

Organization of the hupDEAB genes within the hydrogenase gene cluster of Anabaena sp. strain PCC7120

A 15-kb DNA fragment containing a cluster of hup genes has been identified and cloned from Anabaena sp. strain PCC7120. These genes are located upstream of the hupL gene in the adjacent fragment in the Anabaena chromosome. Sequence analysis of a 3.5-kb HindIII fragment showed the sequence of hupEAB and a part of the hupD gene, all of which showed high sequence similarity with hyp genes of Escherichia coli and hup genes of several nitrogen-fixing bacteria. These genes are oriented in one direction, as are the hup genes of other organisms. Although the Anabaena hupDEAB genes are in the same cluster as the hypABCDE cluster of E. coli, the relative positions of the genes differ and there is no hupC in Anabaena on either side of hupA or hupB. Unlike several other organisms, hupD and hupE are not closely linked or translationally coupled in Anabaena, but are separated by an intergenic space of 453 bp. RT-PCR analysis of RNA obtained from vegetative cells and heterocysts of Anabaena showed that the hupB gene is expressed only in heterocyst-induced cultures. This revised version was published online in June 2006 with corrections to the Cover Date.