Under batch enrichment conditions the substrate is in excess and hence selection is based solely on the maximum specific growth rate of the organisms present (Jannasch, 1967; Parkes, 1982). The organisms selected are characteristically capable of high growth rates at high substrate concentrations, that is, they are 2ijmo<jenous.
The enrichment techniques resulted in the isolation of a number of bacteria capable of rapid and effective growth on the halogenated substrates. This growth was much more efficient than that of organisms isolated directly from the same soil. Apparently the period of mixed
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culture growth in the enrichment cultures selected organisms which were more capable of utilizing the substrate than the parental strains used to inoculate the cultures originally.
It is possible that the presence of a mixed population of micro organisms as opposed to the 'pure cultures' on selective media, represented by the individual organisms and eventually individual colonies, provided the necessary additional metabolites to overcome either the toxic nature of the substrate or the gradual depletion of growth factors in the enrichment medium. The growth of larger populations under conditions not suitable for growth of individual organisms was described by Jannasch (1967b).
Hall and Zuzel (1980) investigated mutations of the ebg A gene of E sch e ric h ia c o l i and discovered that the mutations fall into two classes neither of which could utilize galactosylarabinose. However, when both classes of mutation were present in the same ebg A gene, the ebg enzyme acquired a specificity for this substrate. They suggested that their results showed that a new enzymatic function could evolve via recombination within the ebg A gene. They envisaged the situation in which mutants of the same gene, which had diverged under different selection pressures (Section 1.5), could recombine, when the population was remixed,to generate a new allele with a substrate specificity present
in neither parent. Thus, the daughter organisms could exploit resources, in this case a carbon source, unavailable to either parent.
Work with Pseudomonas putida strain PP3 (Slater e t al„ 1979)
has indicated that the dehalogenases are the growth-rate limiting enzymes. Thus the selection of organisms capable of more efficient utilization
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of the halogenated substrates during the batch enrichment stage suggests that the concentration of dehalogenase within these more efficient organisms was increased, so allowing more rapid growth. This could have been achieved by either selecting for constitutive mutants, or selecting for mutants with gene multiplication (Section 1.4.5) or selecting for strains with more than one different dehalogenase obtained through the activity of any gene transfer mechanism.
The increased growth-rate cannot be explained by the selection of constitutive mutants, as all the dehalogenase mechanisms in the isolates were demonstrated to be inducible. Their activities were absent in cultures grown on succinate as the sole carbon source yet when transferred back to halogenated substrate containing medium activity could be detected.
The apparent requirement for a period of mixed culture growth,in order to isolate bacteria capable of efficient growth on the halo-compounds,
indicates that the selection process could involve the accumulation of different dehalogenase genes in the same organism from different parents. That is by a mechanism similar to the one envisaged by Hall and Zuzel (1980).
The inability of those isolates selected for growth on the halogenated substrates directly from soil samples to grow reliably on these compounds caused practical difficulties which culminated in a decision to concentrate further experimental effort towards the study of the bacteria isolated from the enrichment cultures (the E isolates). These organisms grew effectively in batch culture with MCA or 2MCPA as their sole carbon source. However, DCA was apparently relatively toxic to all the isolates, including those enriched with DCA as the carbon source, and growth on 22DCPA was restricted. Because of this further research centred around the following
of the halogenated substrates during the batch enrichment stage suggests that the concentration of dehalogenase within these more efficient organisms was increased, so allowing more rapid growth. This could have been achieved by either selecting for constitutive mutants, or selecting for mutants with gene multiplication (Section 1.4.5) or selecting for strains with more than one different dehalogenase obtained through the activity of any gene transfer mechanism.
The increased growth-rate cannot be explained by the selection of constitutive mutants, as all the dehalogenase mechanisms in the isolates were demonstrated to be inducible. Their activities were absent in cultures grown on succinate as the sole carbon source yet when transferred back to halogenated substrate containing medium activity could be detected.
The apparent requirement for a period of mixed culture growth,in order to isolate bacteria capable of efficient growth on the halo-compounds, indicates that the selection process could involve the accumulation of different dehalogenase genes in the same organism from different parents. That is by a mechanism similar to the one envisaged by Hall and Zuzel (1980).
The inability of those isolates selected for growth on the halogenated substrates directly from soil samples to grow reliably on these compounds caused practical difficulties which culminated in a decision to concentrate further experimental effort towards the study of the bacteria isolated from the enrichment cultures (the E isolates). These organisms grew effectively in batch culture with MCA or 2MCPA as their sole carbon source. However, DCA was apparently relatively toxic to all the isolates, including those enriched with DCA as the carbon source, and growth on 22DCPA was restricted. Because of this further research centred around the following
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isolates: Pseudomonas sp. strain E2; Pseudomonas sp. strains E3,
E4 and E6, all of which utilized 2MCPA as their sole carbon and energy source, and Alaaligenes sp. strains E20 and E22. The former Aloaligenes
strain although initially capable of growth on MCA in batch culture, later became unable to do so yet it maintained the ability to utilize this compound in continuous-flow culture. Aloaligenes sp. strain E22,
originally isolated from the MCA enrichment culture, later apparently lost the ability to grow on MCA in both batch and continucus-flow culture, yet was capable of growth on 2MCPA under either of the culture conditions. The reason for these changes is not understood but
apparently MCA became toxic to these organisms such that even at 10 and 50-fold lower concentrations, that is 0.05 and 0.01 gC MCA jf1, growth was inhibited.
The results from the identification studies (Table 3.4) indicated a different result for the isolate E6 and isolate E60. The latter was from a stock culture of the other. The isolate E6 was tentatively identified as Pseudomonas maltophilia largely on its auxotrophic
requirement for methionine, a requirement which isolate E60did not exhibit. As they are known to be the same organism it is suggested that the isolate E6 should have been grouped along with isolates E3 and E4 and described as a Pseudomonas sp. especially as nearly all
of the characteristics of isolate E6 are the same as isolates E3 and E4.
Although isolates E20 and E22 were classed as strains of Aloaligenes
sp. it was suggested that with different tests for glucose utilization (Tatum e t al. ,1974) both isolates may have been classed as Aahromobaater
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CHAPTER 4
DEHALOGENASE COMPLEMENT UNDER BATCH-CULTURE CONDITIONS
Considerable variation has been recorded in the catalytic- activity of several dehalogenases from a number of species of micro organisms (Davies & Evans, 1962; Little & Williams, 1971; Goldman
et al., 1968; Berry et al., 1979; Slater et al., 1979). These enzymes
demonstrate different substrate specificities; for example, Jensen (1960) noted that the MCA-induced dehalogenase of Pseudomorias dehalogenana
attacked MCA and DCA, when the organism belonged to groups I or II, but if a group III organism, the substrate range was increased to include 22DCPA. Several investigations on different bacterial strains (Goldman et al., 1968; Weightman et al., 1979; Weightman & Slater,
1980) have suggested that more than one dehalogenase was present. This was also shown for the fungu*Triahodema viride (Jensen, 1960).
Although a number of dehalogenase systems have been described, dehalogenase variation has not been systematically studied. The dehalogenase activities of soil bacteria, isolated by batch-enrichment, have been investigated and the enzyme profiles studied using poly acrylamide gel electrophoresis.