BIOCHEMICAL STUDIES OF A ZYMOMONAS MOBILIS MUTANT WITH DELAYED GROWTH ON HIGH GLUCOSE CONCENTRATIONS

BIOCHEMICAL STUDIES OF A ZYMOMONAS MOBILIS MUTANT WITH DELAYED GROWTH ON HIGH GLUCOSE CONCENTRATIONS

M. PAPAMICHAEL^*, A-I. KOUKKOU, E. DOUKA, G. VARTHOLOMATOS, K. A. MASAVETAS^#, AND C. DRAINAS

University of Ioannina, Department of Chemistry, Ioannina – 451-10, GREECE.

^# National Technical University, Department of Chemical Engineering, Section of Materials Science and Engineering, Lab. of Physical Chemistry, Iroon Polytechniou 9 Zografou, Athens - 157 80, GREECE.

^*To whom correspondence should be addressed: University of Ioannina, Dept. of Chemistry, Sector of Organic Chemistry & Biochemistry, 451-10 Ioannina, GREECE. Tel.: +30 651 98395, FAX: +30 651 47832, E-Mail address: epapamic@cc.uoi.gr

ABSTRACT

When experimental data dealing with binding of ligands to proteins are exhibited biphasic kinetics in Eadie – Hofstee plots, one cannot discriminate if a two-components system having one binding site per molecule, or a one-component system having two binding sites per molecule exist. A novel diagnostic test has been developed in this work, which can be used to discriminate between these cases, based on the determination of the fractal dimension of the Michaelis-Menten equation.

Key words: Zymomonas mobilis, Glucose uptake, Biphasic plots, Fractal dimension, Diagnostic test

INTRODUCTION

In a relatively recent paper (1) we investigated the ability of Zymomonas mobilis to grow on elevated glucose concentrations by the use of a derivative of strain ATCC 10988, the CU1Rif2. This mutant showed more than 20 hours lag period when transferred from a 0.11 M to 0.55 M glucose liquid medium, whereas, on a 0.55 M glucose solid medium it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable or non-fermentable sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild type cells had been incubated for 3 hours and removed at the beginning of their exponential phase. This preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Heating or protease treatment impaired non-delayed growth of CU1Rif2 cells on 0.55 M glucose previously conditioned by the wild type. This result indicates that a putative factor produced by the wild type, and formed during the lag period at a time much shorter as compared to CU1Rif2 mutant, is excreted in the growth medium allowing initiation of growth at elevated glucose concentrations. Accordingly, we suggested that in Zymomonas mobilis a diffusible heat-labile proteinaceous factor, transitionally not present in 0.55 M glucose of CU1Rif2 cultures, triggers growth on 0.55 M glucose. The basis of this phenomenon has not been studied before in Zymomonas mobilis.

Here we present new results in order to elucidate particular features of this diffusible proteinaceous factor as a glucose transporter. In this context, we applied a conventional methodology, by using the strain ATCC 10988 of Zymomonas mobilis as well as its CU1Rif2 mutant, and we collected a series of glucose uptake experimental data, which were analyzed kinetically by the application of a novel diagnostic test.