The effect of growth temperature on the loss of virulence of the fish pathogen Aeromonas salmonicida was investigated. Three virulent strains were grown in Trypticase soy broth at temperatures ranging from 22 to 30 degrees C. Growth at a higher-than-optimal temperature (26 to 27 degrees C for the three strains studied) resulted in the selection of spontaneous attenuated derivatives in the initial bacterial population. For example, virulent bacteria represented less than 10% of the population of a culture grown at 30 degrees C, and attenuated derivatives were easily isolated by streaking the culture on solid medium and picking single colonies. Virulent strains autoaggregated during growth and possessed a cell wall layer (A-layer) external to the outer membrane, as previously described. Attenuated strains did not autoaggregate and did not possess the A-layer. The A-layer apparently shielded bacteriophage receptors and a mannose-specific yeast agglutinin located in the outer membrane. Thus, virulent strains exhibited impaired adsorption of phages, whereas attenuated strains were phage sensitive. Furthermore, attenuated strains agglutinated yeast cells but virulent strains did not. The attenuated strains had higher maximum growth temperatures than their virulent parent strains, and this accounts for their selection at high temperatures. It is proposed that the A-layer contributes significantly to the physical properties of the A. salmonicida cell envelope and that these physical properties of the A. salmonicida cell envelope and that these physical change upon loss of the A-layer to permit growth at a higher-than-usual temperature.

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives.

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Two types of mutants which displayed altered regulation of the NAH catabolic plasmid-encoded catechol meta-cleavage pathway were isolated in Pseudomonas putida. Altered metabolic control was indicated by assay of catechol 2,3-dioxygenase. In one type of mutant the catechol 2,3-dioxygenase was synthesized constitutively. In the other type the range of carbon sources which induce the catechol 2,3-dioxygenase was increased.