Mutant strains obtained after genome shuffling show substantial improvement in the ability to grow in the presence of PCP on solid medium. Several strains obtained after the third round of shuffling can grow on plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of concentrations higher than 0.6 mM. Characterization of selected mutant strains in liquid medium shows that the improvement in the ability to grow in the presence of PCP is correlated with increased degradation of PCP and so is not simply due to mutations that result in exclusion of PCP from the cell.
A clue to the mechanisms underlying the improved performance of the mutant strains is provided by the data in Fig. , which demonstrate that the growth rate of the mutant strains is closely correlated with the ability to remove PCP from the medium. Thus, an important factor in the rate of PCP degradation is simply the cell mass that can be achieved. The enhanced growth rate of the mutant strains might be due to mutations that allow the cells to use the nutrients available in one-quarter-strength TSB more effectively. Indeed, the mutant strains grow faster than the wild type on one-quarter-strength TSB, even in the absence of PCP. However, mutations that allow faster removal of PCP or that ameliorate the adverse effects of PCP or its metabolites must also be involved, since the mutant strains were able to grow in the presence of high levels of PCP that completely inhibited the growth of the original strain.
Constitutive expression of pcpB
, and pcpD
was found in four of the mutant strains. These strains degraded PCP most quickly when the cells were not preinduced with PCP (Fig. ). Constitutive expression of PCP degradation genes may protect cells from the initial shock of exposure to PCP by allowing degradation to begin immediately. Expression of the PCP degradation genes is regulated by pcpR
, a LysR family transcriptional regulator (2
). LysR transcriptional regulators appear to function as tetramers (11
) that bind to promoter DNA and induce a sharp bend of about 78° (12
). Upon binding of inducer to the regulatory domain, the angle is relaxed to about 54° (12
) and transcription is activated. Previous studies of LysR transcriptional regulators have shown that inducer-independent expression can result from mutations in the regulatory domain of the protein (7
). However, we found no mutations in pcpR
in any of the strains, including those that constitutively express pcpB
, and pcpA
. It is possible that mutations in the promoter regions upstream of pcpB
might allow recognition by another transcriptional regulator capable of directing the expression of these genes in the absence of PCP. This possibility can be ruled out because the promoter region upstream of pcpB
was not mutated in any of the strains. Thus, the most likely explanation is that a mutation in a gene encoding another transcriptional regulator allowed it to bind to the promoter regions of pcpB
and direct expression of the PCP degradation genes.
Strains containing mutations leading to constitutive expression of PCP degradation genes did not necessarily perform better when the cells were pretreated with PCP (Fig. ), suggesting that other types of mutations also conferred enhanced degradative abilities. An obvious mechanism by which PCP degradation might be improved would be up-regulation of the enzymes in the metabolic pathway. Surprisingly, analysis of crude extracts showed that there were no discernible differences in the pattern of proteins produced by the wild-type and mutant strains. Furthermore, assays of PCP hydroxylase and TCBQ reductase in crude extracts of cells grown in the presence of PCP showed no significant differences between the wild-type and mutant strains, with a single exception. The PCP hydroxylase activity in strain 206 was only 63% of that of the wild-type strain. The gene encoding PCP hydroxylase in this strain was sequenced and found to have mutations resulting in changes of Arg45 and Arg74 to Cys. On the basis of sequence comparisons with the homologous and structurally characterized phenol hydroxylase (6
), Arg45 corresponds to a residue involved in binding flavin (Lys43), while Arg74 corresponds to a residue located at the subunit interface (Lys72). Mutation of either or both of these residues could lead to the observed diminution of activity in the strain 206 enzyme.
We suspect that multiple mutations are responsible for the improved phenotypes exhibited by the mutant strains, as the performance of the mutants increased with successive rounds of shuffling, presumably as a result of the combination of different beneficial mutations. Support for the hypothesis that the improved phenotypes were achieved by genome shuffling rather than by accumulation of successive mutations within strains is provided by control experiments in which the initial mutant population was replated without protoplast fusion. No clones capable of growth on 2 mM PCP were found, while plating of a comparable number of cells after protoplast fusion gave 50 colonies on plates containing 2 mM PCP.
Taken together, the different rates of growth and PCP degradation among the mutants when they are grown under different conditions (compare Fig. and ), the differences in the regulation of the PCP degradation genes (Fig. ), and the mutations in pcpB and pcpD (Table ) demonstrate that the seven mutant strains are genetically distinct. This finding suggests that improved performance can be achieved in a number of different ways. The mutations leading to improved performance appear to confer enhanced growth under the selection conditions and enhanced resistance to the toxicity of PCP and TCBQ. Notably, mutations leading to higher activities of the first two enzymes in the pathway, which limit flux through the pathway and detoxify a highly toxic intermediate, respectively, were not found. This is not surprising, since the experimental design involves initial selection for mutants that have improved growth in the presence of PCP. Mutations resulting in substantial improvement of PCP hydroxylase activity would likely result in decrease fitness because of the increased production of toxic TCBQ. Furthermore, if the existing TCBQ reductase activity is already sufficient to protect the cells from the toxic effects of the small amount of TCBQ formed by PCP hydroxylase, then substantial improvements in this activity would also not enhance fitness. Thus, the initial pool of mutants that displayed improved growth on plates containing PCP would not be expected to include mutants with improved versions of either PCP hydroxylase or TCBQ reductase.
The findings reported here have important implications. It is remarkable how much improvement in PCP degradation could be achieved without altering the key enzymes in the degradative pathway. These improvements apparently resulted from mutations that enhanced growth under these particular growth conditions and enhanced resistance to the toxicity of PCP. Thus, we expect that genome shuffling procedures should be most successful when they are tailored for particular applications by matching the selection conditions to the conditions of the process for which the bacteria will be used. In addition, these results suggest that genome shuffling will provide an excellent experimental tool for examination of fitness landscapes by generating a number of mutant strains that have reached the same phenotype via different routes.