A study of extrachromosomal elements and gene transfer in soil bacteria

Abstract

In an attempt to investigate the role of bacterial extrachromosomal genetic elements (bacteriophages and plasmids), and processes of gene transfer in the adaptation of soil bacteria to their environment, an investigation was carried out into the ecology and transferability of extrachromosomal elements (ECEs) in soil bacteria.

Preliminary experiments served to demonstrate the prevalence of ECEs by examining bacteriophages from the genus Bacillus, and plasmid-related markers in a variety of soil bacteria. As a result of these studies the property of mercury resistance (Hg r) was focussed upon in order to provide an easily monitored marker for use in gene transfer experiments.

A diverse population of bacteria, all with the common property of resistance to mercuric ions (Hg²⁺), was isolated from soils with no known history of mercury pollution. Each isolate was taxonomically classified, and screened for its resistance to a variety of antibiotics and heavy metals as well as organomercurial compounds. Bacillus and Pseudomonas were the predominant genera within the mercury resistant population. From the resistance properties of the Hg r organisms there was no convincing evidence of consistent linkage between the common mercury marker, and other antimicrobial agents, although several isolates were also tolerant of organomercurial and arsenic compounds. Treatment with ethidium bromide successfully cured several Bacillus strains of mercury resistance, and the instability of the Hg r phenotype in some other isolates was consistent with the view that the genes responsible were located on extrachromosomal elements.

Seventy-six distinct mercury-resistant isolates were tested in studies to examine gene transmissibility. Of these, four were able to transfer the Hg r phenotype at environmental temperatures, and two harboured plasmids with broad host ranges. As most isolates did not detectably transfer the Hg r property, attempts were made to mobilise the Hg r genes using the wide host range Inc P plasmid RPI. Thirty-five gram negative bacteria accepted RPI and mobilisation experiments resulted in the cotransfer of RP1 markers and resistance to Hg²⁺ in nine cases. Transduction with phage PI in an E. coli K12 background showed that seven of these transfers were the result of the formation of RP1-Hg r or RP1-Hg r-PMA r cointegrates. From three of these cointegrates the transposable nature of the Hg r genes has been confirmed, including one example in which the transposon determines both inorganic and organic mercury resistance.

The observations made in this thesis suggest that phage and plasmid mediated gene transfers are important in the adaptation of soil bacteria to their surroundings, and provide the basis for further studies of gene transfer in the soil environment.