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 Planktothrix 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.

Wild-type strains of the cyanobacterium Planktothrix rubescens have a cluster of gas vesicle (gvp) genes with repeats of alternating gvpA and gvpC. The gvpC occurs in three length variants, all with the same 3'-sequence, OmegaC. Spontaneous non-buoyant mutants had lost some of the alternating gvpAC copies and their gvpC genes had a novel 3'-end sequence, PsiC; additional gvpC genes terminating in this sequence were also found in the wild-type and representatives of other GV genotypes. Alleles of gvpC terminating in PsiC occurred only at the downstream ends of the gvpAC clusters investigated; all other gvpCs terminated in OmegaC. Mutants of strains with the GV3 genotype produced only 30-50% of the gas vesicles present in the wild-type; their gas vesicles had lower mean critical pressures (0.70-0.78 MPa) than those in the wild-type (1.05-1.10 MPa).

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.

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.

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.

To investigate the role of the buoyancy provided by gas vesicles in the facultative anaerobe Halobacterium salinarium PHH1, the growth of a gas-vacuolate (Gv(+)) strain in competition with two gas-vesicle-defective (Gv(def)) mutants was examined. The Gv(+) strain synthesized gas vesicles throughout ifs growth cycle, and floated up to form a thick surface scum during the exponential growth phase in static culture. Mutant Gv(def1) produced significantly fewer gas vesicles than the Gv(+) strain in corresponding stages of growth, although in late stationary phase a small proportion of cells floated up to the surface of static cultures. Mutant Gv(def2) had much lower gas vesicle content in shaken culture and produced negligible amounts of gas vesicles in static culture. The Gv(+) and the two Gv(def) strains grew equally well in shaken cultures, but in static cultures, where steep vertical gradients of oxygen concentration were established, Gv(def1) was outgrown by the Gv(+) strain. Gv(def2) outcompeted the Gv(+) strain in shallow static cultures, perhaps because Gv(def2) carried a smaller protein burden, which offset the benefits of buoyancy. This selection for Gv(def2) was lost in deeper static cultures, although it could be restored by aerating static cultures from below. The results support the hypothesis that the role of buoyancy in halobacteria is to maintain cells at the more aerated surface of brine pools.

Computer simulations of Schottky barrier diodes show that electron heating occurs at high frequency as the depletion edge velocity increases. Mixer characteristics are reappraised in this light.