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This study was carried out as part of a contract funded by the Scottish Office to determine whether approaches to risk assessment, based on current recommendations, were adequate in determining the possibility of an adverse environmental effect of introduction of GMOs to the environment. The studies used a representative soil bacterium, Pseudomonas fluorescens, genetically modified by either chromosomal (FAC510) or plasmid (pUCD607) insertion of lux genes for bioluminescence. The starvation state is considered an important stress factor that bacteria added to soil may face. The ability to recover from this state, when substrates and nutrients become available, may be considered essential for the success of an introduced organism, and may ultimately determine the ecological effects of a GMO. The recovery response to the GMO constructs from carbon starvation was investigated in pure culture studies, and upon addition of starved cells to sterile soil. Pure culture studies clearly demonstrated a starvation-survival disadvantage of both the GMO constructs, as determined by growth and respiratory activity (dehydrogenase activity) when resuscitated from periods of starvation of up to 21 days. Correlation between dehydrogenase and luminescence activity was investigated. The lack of correlation between the two processes may have pointed to a change in maintenance energy costs as a consequence of genetic modification. Determination of the starvation-survival response in sterile soil was inconclusive, due to high variability and limitations to, and uncertainty in, cell extraction and culture.

The potential role in pathogenicity of extracellular products (ECP) of the fish pathogen Aeromonas salmonicida was investigated. Evidence is presented that determinants of host damage are produced extracellularly by A. salmonicida. In vitro, the bacterium produced a number of extracellular enzymic and ctyolytic activities, including proteases and an heterogeneous haemolysin which was active against rainbow trout erythrocytes. Solubilization of trout erythrocytes in vitro resulted from the cooperative effects of the haemolysin and a caseinolytic protease. The caseinolytic protease was implicated in host damage; the in vivo effects of the caseinase may have been potentiated by haemolysin. The production of extracellular proteolytic and haemolytic activities by a number of isolates of A. salmonicida was studied; some potentially important differences between isolates were found. Evidence is presented that rainbow trout possess humoral mechanisms of resisting the toxic products of A. salmonicida. Proteolytic and haemolytic activities of ECP were inhibited, and the in vivo toxicity of ECP was neutralized, by normal trout serum. In vitro, factors in normal trout serum appearaed to form soluble complexes, as well as insoluble precipitates, with component(s) of ECP. In vitro, ECP caused a reduction in circulating levels of an alpha-2 macroglobulin analogue in rainbow trout. As putative determinants of pathogenicity, extracellular factors of A. salmonicida are potentially important in conferring protective immunity against furunculosis. The humoral immune response of rainbow trout and rabbits to components of ECP was investigated. At least 15 components of ECP, including the caseinase and haemolysin, were immunogenic in the rabbit. In the trout, antibodies to only 4 components of ECP were detected and no antibody to the caseinase or haemolysin was found. It is suggested that the effectiveness of furunculosis vaccines might be improved if the immunogenicity in trout of certain ECP components could be enhanced.

The OXA-2 beta-lactamase gene was first found on a conjugative plasmid R46 from a clinical isolate of Salmonella typhimurium. To test the expression and secretion of OXA-2 beta-lactamase in Streptomyces lividans a shuttle plasmid (pSU101) was created by fusing an Escherichia coli plasmid (pSU8) carrying the OXA-2 beta-lactamase gene with the S. lividans vector pLJ61. The OXA-2 beta-lactamase gene specified by the hybrid plasmid PSU101 was expressed in S. lividans, although at a lower level than in coli. Almost all the beta-lactamase activity was found in the culture supernatant of S. lividans, whereas in E. coli the enzyme was almost wholly cell associated. The identity of the enzyme was established by substrate specificity and isoelectric focusing. The stability and integrity of the plasmid pSU101 in both E. coli and lividans was determined, in comparison with that of pSU8 and pLJ61 plasmids. The promoter regions of the OXA-2 beta-lactamase gene were identified by using promoter-probe plasmid vectors; and by S1 mapping of the transcriptional start-sites coupled to the DNA sequencing of the OXA-2 beta-lactamase gene. Multiple transcriptional start sites were found in both hosts, with the origin of transcription apparently different in the two organisms. Part of this work has been published as a scientific paper which is appended.