CONCLUSIONES Y RECOMENDACIONES

mechanisms (Ornston & Parke, 1976). The catabolism of aromatic compounds has also been used by Dagley (1971) in studying relationships in

Pseudomonas strains, and Moraxella oalooaaetioa and demonstrated

entirely different control mechanisms in the different genera. The regulatory controls in the three Pseudomonas spp.studied were very

similar.

1.4 THE EVOLUTION OF BACTERIAL ENZYME SYSTEMS

A great deal of experimental data on evolution of enzyme systems was presented by Hegeman and Rosenberg (1970),and Markert et al. (1975)

used the lactate dehydrogenase isoenzymic system as a model of the evolution of gene structure, function and regulation.

1.4.1 Retroevolution

The earliest theory proposed to account for the evolution of biosynthetic pathways was that of Horowitz (1945). The hypothesis of retrograde evolution suggested the step-wise and sequential

recruitment of enzymes in reverse order and depended on the assumption that each intermediate of the backwardly evolving pathway was readily available in the prebiotic environment. If the pathway evolved from the beginning it would not be selectively advantageous until the

whole pathway was complete. However if evolution of the pathway occurred in reverse, one step at a time towards the initial reaction, then each mutation would confer a selective advantage since a new, useable end product would be available for growth (Horowitz, 1965). Major problems with this theory concern the extreme lability of many of the intermediates as well as the barrier to their transport in the absence of specialised

b«

transport systems. The evolution of catabolic pathways, cannoyaccounted for by this hypothesis (Jensen, 1976).

1.4.2 Gene duplication

Horowitz (1965) evoked the theory of E.B. Lewis, that the origin of new genetic material involved tandem duplication followed by

functional differentiation. Many aspects of gene duplication, including the mechanisms involved have been discussed by Ohno (1970). New

genetic material is created by gene duplication by a variety of mechanisms for example transposition to give a second copy of the gene, tandem duplications due to unequal crossing over or mini-insertion-like duplications (Cullum & Saedler, 1981) or frame shift mutations (Ohno, 1970). One of the resulting genes is redundant and 1s thus free of selectional pressures which prevented the parental gene from changing.

60

A mutation resulting in the change of a residue directly involved in catalysis would cause loss of function, which in most cases would always be lethal (Watts & Watts, 1968ai; Kimura & Ohta, 1974). Whilst one of the daughter genes provides a viable phenotype, the other provides genetic material which can undergo mutation to produce new enzymes, with different capacities, although they are initially related to the

original gene. Under different environmental conditions the mutated gene product may again become active and provide the organism with a new gene product.

The existence of silent or cryptic genes has been demonstrated in a number of cases. Campbell et al. (1973) deleted the lac 1 for

8-galactosidase in Escherichia coli strain K-12 and selected for

reacquisition of activity and found a new enzyme which possessed different kinetic properties to the lac 1 product. The new gene, ebg, demonstrated

no close ancestory to the lac Z gene and no evidence has been found to

show 0-galactosidase polymorphism in E. coli under normal circumstances.

The ebg locus may represent a silent gene or a multistep mutation of a

gene to produce the new enzyme to hydrolyse lactose. Syvanen and Roth (1972) demonstrated the redundancy of a second ornithine transcarbamylase gene in E. coli K-12 separated from the active gene by 14 min on the E. coli genome map. Schaefler and Maldny (1969) investigated the

expressed and cryptic phospho-B-glucosidases in Enterobacteriaceae and suggested their use in studying evolutionary relationships. The position of duplicate genes relative to each other would vary according to the method of duplication (Watts & Watts, 1968b). Duplication can occur either by small changes in segments of the chromosome or by duplication of the whole chromosome, polypioidization in higher plants

61

represents a form of duplication. Zipkas and Riley (1975) proposed that the genome of E . o o li underwent two sequential duplications} 90° and 180° relationships between functionally related genes, they believed, were too great to be due to chance alone. Gene pairs giving rise to isoenzymes of malate synthetase, glycerol phosphate dehydrogenase and phosphotransferase system enzymes are located at approximately 90° from each other. Those isoenzymes not possessing this relationship were believed to represent examples of convergent evolution. Sequence analysis of a number of proteins indicated that gene duplications have occurred fairly frequently in evolution.

The ability to perform a new function is unlikely to be associated with a great change in structure as this is more likely to lead to complete loss of function, due to incompatibility with the protein's tertiary structure. Rigby e t a l . (1974)anJWatts and Watts (1968a) cite Annelid phosphagen kinases as such an example. They all have one type of catalytic site but their functions are altered by mutations of amino acids in other parts of the molecule associated with the following: substrate binding, specificity towards the substrate, conformational changes associated with catalytic activities and those amino acids associated with maintaining the tertiary and quarternary structure of the enzyme.

Mutants of Pseudomonas aeruginosa have been isolated which can utilize novel amides which could not support growth of parental strains. Betz e t a l . (1974) showed that this was the result of a number of point mutations in the amidase gene of the bacterial strains.