A naturally occurring mercury-resistance, conjugative plasmid, designated pQM 1, was isolated from a bacterial population on the surface of stones from a river using Pseudomonas aeruginosa as a recipient. This was a narrow-host-range plasmid [IncP-13; 165 MDa; Tra+, Hgr, fluorescein mercuric acetate', merbromin', Phi(E79), UVr] confined to some Pseudomonas spp. It was used to demonstrate transfer between bacteria on stones in laboratory microcosm experiments and in situ. Transfer occurred (3.3 x 10-l to 6.8 x per recipient) at all the temperatures used (6-20°C), although frequencies were lower in the cold. Nutrient status also affected transfer frequency, rich conditions promoting transfer. The presence of competing bacteria in the natural epilithon lowered transfer frequencies, but when unscrubbed stones were heat treated, transfer was enhanced, perhaps because of nutrient release from the heated epilithon.

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log,, molecular size against log,, relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates ( I 3.0 & 1.5 %). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.

In situ mating experiments were done in the River Taff, South Wales, United Kingdom, by using a natural mercury resistance plasmid (pQM1) isolated from a mixture of epilithic bacteria in vitro. The river temperature from March to November was found to influence transfer frequencies strongly (6.8 x 10-9 to 1.5 X 10-2 per recipient). A linear relationship existed between loglo transfer frequency and river temperature (6 to 21°C), a 2.6°C change in temperature giving a 10-fold change in transfer frequency. In vitro experiments showed that pQM1 transferred most efficiently between fluorescent pseudomonads and that one epilithic isolate (Pseudomonasfluorescens) was an efficient donor in situ. Experiments with a P. putida recipient showed that intact epilithic bacterial communities could transfer mercury resistance plasmids in situ at frequencies of up to 3.75 x 10-6 per recipient. Nineteen of the large (>250-kilobase) plasmids isolated by transfer into P. putida were studied in detail and grouped into seven types by restriction digests. Mercury resistance and UV resistance were found to be common linked phenotypes in 19 of the 23 plasmids tested.

The survival of 11 species of Gram-negative and Gram-positive bacteria was examined on different surfaces exposed to desiccation. There were large variations between species; Pseudomonas spp. and Rhizobium leguminosarum biovars survived for less than 2 d, whilst Enterococcus spp. survived for more than 11 weeks. The type of surface on to which the bacteria were deposited affected survival, but with different effects between species. In addition the survival of spontaneous nalidixic acid-resistant (Nal-r) mutants of a natural Escherichia coli isolate were compared. Overall the differences were slight, but of seven resistant mutants, five survived better than the parent whilst one survived less well. Nine transposon insertion derivatives of one of the Nal-r mutants (ECO80) which survived better than the parent were compared; all survived similarly to the parent except ECO883 which survived less well. The growth characteristics of ECO883 and ECO80 were compared; at high osmotic pressures (> 0.4 mol 1-1 NaCl) ECO883 grew more slowly and showed a longer lag time than the parent. Of the osmoregulatory functions studied, ECO883 appeared to be altered with respect to K+ transport or accumulation, although the transposon insertion had occurred in a gene distant from known K+ transport genes.

A method for in situ mating experiments is described which involved overnight incorporation of donors containing the mercury resistance plasmid pQM1 and recipients into the epilithon on separate river stones. The stones were then joined to begin the mating. Transfer frequencies obtained were between 2.2 x 10-' and 2.5 X 10-6 per recipient and appeared to depend on the donor-to-recipient ratio (489/1 to 0.0047/1) and not on the river temperature (12 to 19°C). Controls showed that the low density of donors and recipients at the end of the experiment (3.4 x 102 to 7.0 x 105 cm-2) did not significantly affect the heterotrophic bacterial count (1.43 x 106 to 6.39 x 106 cm-2) nor the fluorescent-pseudomonad count (2.3 x 104 to 9.33 x 104 Cm-2).