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Biomedical subjects

A E Walsby

Publications and source records attributed to A E Walsby.

At least 19 recordsLinked to original sources
PubMed2002

Genetic diversity within populations of cyanobacteria assessed by analysis of single filaments.

We have developed a technique for determining the genetic structure of populations of filamentous cyanobacteria. The sequence diversity at specific gene loci is first characterised in a range of clonal cultures; subsequent analysis involves individual trichomes collected directly from natural populations. This technique has been used to examine the population genetic structure of Nodularia in the Baltic Sea and Planktothrix in Lake Zürich. For Nodularia, studies utilising four polymorphic loci reveal that even though there is a degree of linkage disequilibrium, horizontal transfer of genetic information has been sufficient to generate many of the possible allelic combinations. Analyses reveal both spatial and temporal variation in population genetic structure. Other studies of both Nodularia and Planktothrir have shown a correlation between particular alleles at the gvpC locus and the critical pressure of the gas vesicles that accumulate within the cell. We are now investigating how the natural selection of different gas vesicle phenotypes, imposed by changes in the depth of the upper mixed layer of the water column, affects the relative success of individual cyanobacteria possessing different gvpC alleles.

Archaeal Proteins↗
PubMed2000

Gas vesicle genes in Planktothrix spp. from Nordic lakes: strains with weak gas vesicles possess a longer variant of gvpC.

In cyanobacteria of the genus Planktothrix:, there are three length variants of gvpC, the gene that encodes the outer protein of the gas vesicle. Sequence analyses indicated that the three allelic variants of gvpC differ principally in the presence or absence of a 99 nt and a 213 nt section. Strains with the new variant, gvpC(28), which encodes a 28 kDa form of GvpC, produce gas vesicles that collapse at the relatively low critical pressure (p(c)) of 0.61-0.75 MPa. The authors have identified 12 classes of gvp genotypes that differ in the number and arrangement of alternating gvpA-gvpC genes and in the presence of OmegaC, a fragment of gvpC. The gvpC(28) gene was found to be the most common variant of gvpC amongst 71 strains of Planktothrix: isolated from Nordic lakes: 34 strains contained only gvpC(28); 22 strains, which possessed only the shorter gvpC(20) gene, produced gas vesicles with a higher p(c) of 0.76-0.91 MPa; and 15 strains, which possessed both gvpC(20) and gvpC(28), also produced the stronger gas vesicles. Genotypes with only the gvpC(28) genes were more common amongst green Planktothrix: strains (33 out of 38) than red strains (one out of 33). It is suggested that there is competition between the strains producing the two types of gas vesicles, with the stronger forms favoured in lakes deeper than 60 m, in which the combination of cell turgor pressure and hydrostatic pressure can collapse the weaker gas vesicles. The fact that none of the Nordic lakes are deeper than 67 m would explain the absence of the gvpC(16)-containing strains that produce even narrower gas vesicles of p(c) 1.0-1.2 MPa, which are common in the much deeper Lake Zürich.

Alleles↗
PubMed1999

The diversity of gas vesicle genes in Planktothrix rubescens from Lake Zürich.

Part of the gas vesicle gene cluster was amplified by PCR from three strains of Planktothrix rubescens isolated from Lake Zürich, Switzerland. Each contains multiple alternating copies of gvpA and gvpC. All of the gvpA sequences in the different strains are identical. There are two types of gvpC: gvpC20, of length 516 bp, encodes a 20 kDa protein of 172 amino acid residues (whose N-terminal amino acid sequence is homologous with the sequence of GvpC in Planktothrix [Oscillatoria] agardhii); gvpC16, of length 417 bp, encodes a 16 kDa protein of 139 amino acid residues that differs in lacking an internal 33-residue section. An untranslated 72 bp fragment from the 3' end of gvpC, designated omegaC, is also present in some strains. The two types of gvpC and presence of omegaC could be distinguished by the different lengths of PCR amplification products obtained using pairs of oligonucleotide primers homologous to internal sequences in gvpC and gvpA. Three genotype classes were found: GV1, containing only gvpC20; GV2, containing gvpC20 and omegaC; and GV3, containing gvpC16, gvpC20 and omegaC. Subclasses of GV2 and GV3 contained either one or two copies of omegaC. The accompanying paper by D. I. Bright & A. E. Walsby (Microbiology 145, 2769-2775) shows that strains of the GV3 genotype produce gas vesicles with a higher critical pressure than those of GV1 and GV2. A PCR survey of 185 clonal cultures of P. rubescens isolated from Lake Zürich revealed that 3 isolates were of genotype GV1, 73 were of GV2 and 109 were of GV3. The PCR technique was used to distinguish the gas vesicle genotype, and thence the associated critical-pressure phenotype, of single filaments selected from lakewater samples. Sequence analysis of the 16S rDNA and of regions within the operons encoding phycoerythrin, phycocyanin and Rubisco confirmed that these strains of Planktothrix form a tight phylogenetic group.

Amino Acid Sequence↗
PubMed1999

The relationship between critical pressure and width of gas vesicles in isolates of Planktothrix rubescens from Lake Zürich.

The mean critical collapse pressure (p(c)) of gas vesicles in 81 strains of the cyanobacterium Planktothrix rubescens from Lake Zürich, Switzerland, was bimodally distributed between a minimum of 0.86 MPa and a maximum of 1.17 MPa. Measurements were made of the cylinder diameter (d) of gas vesicles isolated from seven of the strains. The mean diameter, which varied from 48 to 61 nm, was inversely related to p(c), in keeping with the theory of strength of thin-walled rigid cylinders. These measurements extended the range of p(c)-width relationship of gas vesicles, which can be described by the expression p(c) = 461(d/nm)(-1.53) MPa. p(c) was correlated with gas vesicle genotype (see the accompanying paper by S. J. Beard, B. A. Handley, P. K. Hayes & A. E. Walsby, Microbiology 145, 2757-2768): of the 81 strains investigated, all those with the gas vesicle genotype GV2 produced gas vesicles with a mean p(c) of less than 1.0 MPa, whereas those of GV3 had a mean p(c) of greater than 1.0 MPa. It is suggested that gas vesicles of the GV3 strains, which are narrower and stronger than any previously recorded in freshwater cyanobacteria, have evolved to withstand the high hydrostatic pressures during deep winter mixing in Lake Zürich.

Bacterial Proteins↗
PubMed1996

Direct observation of protein secondary structure in gas vesicles by atomic force microscopy.

The protein that forms the gas vesicle in the cyanobacterium Anabaena flos-aquae has been imaged by atomic force microscopy (AFM) under liquid at room temperature. The protein constitutes "ribs" which, stacked together, form the hollow cylindrical tube and conical end caps of the gas vesicle. By operating the microscope in deflection mode, it has been possible to achieve sub-nanometer resolution of the rib structure. The lateral spacing of the ribs was found to be 4.6 +/- 0.1 nm. At higher resolution the ribs are observed to consist of pairs of lines at an angle of approximately 55 degrees to the rib axis, with a repeat distance between each line of 0.57 +/- 0.05 nm along the rib axis. These observed dimensions and periodicities are consistent with those determined from previous x-ray diffraction studies, indicating that the protein is arranged in beta-chains crossing the rib at an angle of 55 degrees to the rib axis. The AFM results confirm the x-ray data and represent the first direct images of a beta-sheet protein secondary structure using this technique. The orientation of the GvpA protein component of the structure and the extent of this protein across the ribs have been established for the first time.

Anabaena↗
PubMed1995

GvpCs with reduced numbers of repeating sequence elements bind to and strengthen cyanobacterial gas vesicles.

We have previously shown that the gas-vesicle protein GvpC is present on the outer surface of the gas vesicle, can be reversibly removed and rebound to the surface, and increases the critical collapse pressure of the gas vesicle. The GvpC molecule, which contains five partially conserved repeats of 33 amino acids (33-RR) sandwiched between 18 N-terminal and 10 C-terminal amino acids, is present in a ratio of 1:25 with the GvpA molecule, which forms the ribs of the gas vesicle. By using recombinant techniques we have now made modified versions of GvpC that contain only the first two, three or four of the 33-amino-acid repeats. All of these proteins bind to and strengthen gas vesicles that have been stripped of their native GvpC. Recombinant proteins containing three or four repeats bind in amounts that give the same ratio of 33-RR:GvpA (i.e. 1:5) as the native protein, and they restore much of the strength of the gas vesicle; the protein containing only two repeats binds at a lower ratio (1:7.7), however, and restores less of the strength. Ancestral proteins with only two, three or four of the 33-amino-acid repeats would have been functional in strengthening the gas vesicle but the progressive increase in number of repeats would have provided strength with increased efficiency.

Amino Acid Sequence↗
PubMed1994

Gas vesicles.

The gas vesicle is a hollow structure made of protein. It usually has the form of a cylindrical tube closed by conical end caps. Gas vesicles occur in five phyla of the Bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. By regulating their relative gas vesicle content aquatic microbes are able to perform vertical migrations. In slowly growing organisms such movements are made more efficiently than by swimming with flagella. The gas vesicle is impermeable to liquid water, but it is highly permeable to gases and is normally filled with air. It is a rigid structure of low compressibility, but it collapses flat under a certain critical pressure and buoyancy is then lost. Gas vesicles in different organisms vary in width, from 45 to > 200 nm; in accordance with engineering principles the narrower ones are stronger (have higher critical pressures) than wide ones, but they contain less gas space per wall volume and are therefore less efficient at providing buoyancy. A survey of gas-vacuolate cyanobacteria reveals that there has been natural selection for gas vesicles of the maximum width permitted by the pressure encountered in the natural environment, which is mainly determined by cell turgor pressure and water depth. Gas vesicle width is genetically determined, perhaps through the amino acid sequence of one of the constituent proteins. Up to 14 genes have been implicated in gas vesicle production, but so far the products of only two have been shown to be present in the gas vesicle: GvpA makes the ribs that form the structure, and GvpC binds to the outside of the ribs and stiffens the structure against collapse. The evolution of the gas vesicle is discussed in relation to the homologies of these proteins.

Amino Acid Sequence↗
PubMed1993

The distribution of the outer gas vesicle protein, GvpC, on the Anabaena gas vesicle, and its ratio to GvpA.

Previous studies have shown that gas vesicles isolated from the cyanobacterium Anabaena flos-aquae contain two types of protein, GvpA, a small hydrophobic protein that forms the main ribbed structure, and GvpC, a protein comprising five repeats of a 33-amino-acid-residue motif, which is located on the outer surface of the GvpA shell. GvpC was shown to increase the critical collapse pressure of the gas vesicles; it was thought to do this by forming a series of molecular ties that bind the ribs together. We now show that antibodies raised against GvpC label both the central cylinders and the conical end caps of native gas vesicles but fail to bind to gas vesicles that have been stripped of GvpC. The molar ratio of GvpA to GvpC has been calculated from amino acid analyses of gas vesicle hydrolysates by reference to the abundance of amino acids that occur predominantly or exclusively in one protein or the other; the molar ratio was found to be 25:1 in freshly isolated gas vesicles and 23:1 in gas vesicles saturated with GvpC. We have considered three ways in which the 33-residue repeats of GvpC might interact with the crystallographic unit cell of GvpA molecules in the ribs. The Anabaena GvpC will bind to and restore the strength of gas vesicles isolated from Aphanizomenon and Microcystis that lack their native GvpC.

Amino Acids↗
PubMed1992

Gas vesicles are strengthened by the outer-surface protein, GvpC.

The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea. Recombinant GvpC was purified from inclusion bodies, produced in an E. coli strain containing an expression vector bearing the gene encoding GvpC from A. flos-aquae, and then solubilised in 6 M urea. This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present. The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value. These results indicate that the function of GvpC is to increase the strength of the structure.

Anabaena↗
PubMed1992

The homologies of gas vesicle proteins.

In addition to GvpA, the main structural protein, an SDS-soluble protein has been found in gas vesicles isolated from six different genera of cyanobacteria. N-terminal sequence analysis of the first 30 to 60 residues of the gel-purified proteins showed that they were homologous to GvpC, a protein that strengthens the gas vesicle in Anabaena flos-aquae. The proteins from some of the organisms showed rather low homology, however, and this may explain why the genes that encode them have not been found by Southern hybridization studies. The gas vesicles of another cyanobacterium, Dactylococcopsis salina, contained two SDS-soluble proteins (M(r) 17,000 and 35,000) that were identical in sequence for the first 24 residues but not thereafter; these two proteins showed no clear homology to GvpC. The sequence of GvpA, the main structural gas vesicle protein, was very similar in each of the organisms investigated. GvpA from the purple bacterium Amoebobacter pendens was different for the first 8 residues but 51 of the next 56 residues were identical to those of the cyanobacterial GvpA. Analysis of the GvpA and GvpC sequences provides support for the idea that the low diversity of GvpA reflects a high degree of conservation rather than a recent origin followed by lateral gene transfer between different bacteria.

Amino Acid Sequence↗
PubMed1991

Antibodies to the N-terminal sequence of GVPa bind to the ends of gas vesicles.

Antibodies were raised against intact gas vesicles of Anabaena flos-aquae, and against a synthetic peptide (GVPaNT) whose sequence is identical to the N-terminal region of the main gas vesicle protein, GVPa. A two-stage centrifugation procedure is described for separating gold-labelled antibodies bound to gas vesicles from unbound antibodies. The GVPaNT antibody bound to gas vesicles that had been previously rinsed with SDS to remove the outer gas vesicle protein, GVPc. Treatment with this antibody caused the gas vesicles to aggregate together end-to-end rather than side-by-side. The binding of the anti-GVPaNT-immunogold particles to the gas vesicle was restricted to the conical ends of the structure. These observations indicate that the sequence to which the GVPaNT antibodies were raised, residues 1 to 13 of the GVPa molecule, is exposed only at the outer surface of the cones and that it is normally obscured by GVPc. As GVPa forms both the conical ends and the cylindrical midsection of the gas vesicle, exposure of the N-terminal sequence only in the cones must be due to differences in the contact between adjacent GVPa molecules in the central cylinders and end-cones.

Amino Acid Sequence↗
PubMed1988

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

Amino Acid Sequence↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1983

Molecular weight of gas-vesicle protein from the planktonic cyanobacterium Anabaena flos-aquae and implications for structure of the vesicle.

The gas vesicle of the planktonic cyanobacterium Anabaena flos-aquae is a cylindrical shell made of protein enclosing a gas-filled space. Protein sequence analysis shows that the vesicle is made from a single protein. By gel electrophoresis and amino acid analysis its molecular weight was estimated to be 20 600. Taken with previously obtained X-ray data, a simple interpretation of its molecular structure is of the polypeptide snaking in six pairs of antiparallel chains, three in each layer. The molecule would repeat along the ribs of the vesicle at intervals of 3.4 nm.

Amino Acid Sequence↗