Biosynthesis of extracellular polysaccharides in Klebsiella aerogenes

Abstract

The cultural characteristics of the capaulate strains K« aerogenes Al and A4 were disoussed, and their mutation to strains which had lost the ability to synthesize exopolysaccharide• The presence of a capsule was shown to protect the micro-organism against dessication, against attaok by organic cations and enzymes degrading the cell wall, and against phage. A mutant of Al was found, A1S1, which produced exopolysaccharide in the form of slime. The exopoly- 14 saccharides were purified and analysed. Incubation of the cells with C - glucose-6-phosphate led to labelling of all the components of the polysaccharides. Total polysaccharide production was estimated during growth, and a comparison was made using washed cell suspensions between the rate of exopolysaccharide synthesis by A1S1 cells grown for various times in different media. The levels of nine enzymes involved in synthesis of the sugar nucleotide precursors of the exopolysaocharides were assayed in strains Al, A131 and A4. Their specific activities were also established in cultures of various ages, or grown at different incubation temperatures, or grown in different media which resulted in a variation in exopolysaccharide synthesis. There was generally little change in the level of any enzyme assayed. Non-mucoid (o) mutants of strains Al, A1S1 and A4 occurred spontaneously at low frequency and this rate was increased by use of mutagens such as 2-aminopurine and acriflavine. Attempts to obtain reversion of these mutants were unsuccessful. There was, however, some evidence that the ability to synthesize the exopoly¬ saccharide was regained in a few Al(o) strains by transduction. Using Al(o), AlSl(o) and A4(o) mutants, the levels of several enzymes synthesizing the nucleotide sugar precursors of the exopolysaccharides were assayed and compared to the parent strains. No enzyme was deficient and generally their specific activities were the same as the parent. A variant of A1 was found which was very unstable, giving rise to mucoid and non-muooid cells with equal frequency, and a mutent of M- produced far more exopolyaaccharide when grown on galactose as carbon source than on glucose. It appeared to be deficient in the enzyme UDPG pyrophosphorylase. A new class of mutants was discovered, the CR mutants, in which the presence of the exopolysaccharide was temperature dependent. It was only synthesized o at incubation temperatures above 30 . There was a second effect on lipopolysaccharide synthesis which led to altered phage sensitivity patterns, to the characteristic crenated appearance of oolonies, and to autoagglutinability in liquid culture at low incubation temperatures. Double mutants, which retained the CR appearance at all incubation temperatures but which had lost the ability to synthesize the exopolysaccharide, were also isolated. The properties of these mutants and the nature of the mutation were discussed. Finally a system was developed in which cell-free synthesis of the AA exopolysaocharide from the labelled nucleotide sugar precursors occurred. Good incorporation of glucose from UDPG into polysaccharide material was obtained. After pre-incubation of the system with UDPG, galactose from UDPGal was incor¬ porated to one-half this extent, and glucuronic acid from UBPG1UA to one-tenth this extent. Chloramphenicol stopped the incorporation of glucose into poly¬ saccharide material. The possibility of lipid-linked intermediates being involved was considered, and the effect of ADPG on the system found.