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.

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.

Some factors affecting the airborne survival of bacteria outdoors.

Airborne survival of two pseudomonads and a reference strain of Escherichia coli (strain MRE 162) was studied outdoors using a modified microthread technique. When cells of E. coli were suspended as clusters, survival was much greater than single cells, particularly outdoors. Culture age had a highly significant effect on survival of Pseudomonas maltophila with survival of 24 h cultures being more than 100-fold higher than 48 h cultures. Survival of Pseudomonas fluorescens was variable and depended also upon the method of culture. Survival of E. coli and Ps. maltophila was studied at three locations differing in air quality and was found to be significantly reduced outdoors, particularly when held in direct daylight. Outdoor survival was not significantly different at the three locations but was reduced at increasing temperatures. There was no apparent effect of wind direction or air quality. Results are discussed with reference to the release of genetically-modified micro-organisms.

The effect of colony age on PCR fingerprinting.

The pattern of DNA fragments produced by the polymerase chain reaction using arbitrary primers is strongly influenced by the age of the colonies sampled. We show that the number, clarity and reproducibility of the bands produced is optimal when very young colonies are sampled.

Some factors affecting airborne survival of Pseudomonas fluorescens indoors.

The effect of relative humidity (RH) on the airborne survival of a Pseudomonas fluorescens strain was studied at 20, 40, 60 and 80% RH indoors. The aero-stable spore of Bacillus subtilis subsp. niger, used as a tracer of physical losses was compared with a light scattering particle counter, as there were doubts about the reliability of the spore as tracer. The Rion counter was validated before use and found to give a good estimate of relative physical losses providing spray suspensions contained between 10(7) and 10(9) cfu ml-1 cells and that the humidity was not more than 80% RH. Pseudomonas fluorescens, strain P+S+, suspended in distilled water survived best at mid humidities and least at 80% RH. When suspended in 1% glycerol there was an apparent 'increase' in viability after an initial rapid reduction. This was thought to be due to delay in equilibration of glycerol in the cell membrane after concentration on dehydration. The cells were thought to be unstable and sensitive to the stress of rehydration before equilibration occurred. The findings are discussed in relation to Cox's theories of outer membrane damage on aerosolization.