Identification, Genetic and Biochemical Analysis of Genes involved in Synthesis of Sugar Nucleotide Precursors of Xanthan Gum

Abstract

A genetic and biochemical analysis of Xanthomonas campestris chromosomal functions required for xanthan polysaccharide synthesis (xps) was undertaken. Seven xps DNA regions were isolated after conjugation of chemically induced non-mucoid mutants with a genomic library of X. campestris DNA. No overlapping segments between regions were detected, based on physical mapping, indicating the unlinked character of these regions. Clones complementing several different mutants belonging to the same region contained overlapping segments of X. campestris chromosomal DNA. Complementation and biochemical analysis, and DNA mapping were used to identify and characterize xpsZZZ, ZV and VZ DNA regions. Mutants in these three regions were able to synthesize both lipid intermediates and xanthan gum in vitro when sugar nucleotides were provided as substrates. HPLC analysis of the intracellular sugar nucleotide content showed that the XpsIII group comprises two different classes of mutants : XpsIIIA, defective in UDP-glucose, UDP-glucuronic acid and GDP-mannose, and XpsIIIB,defective in GDP-mannose. XpsIV mutants were defective in UDP-glucose and UDP-glucuronic acid, and XpsVI mutants were defective only in UDP-glucuronic acid. Analysis of enzyme activities involved in the synthesis of UDP-glucose, GDP-mannose and UDP-glucuronic acid indicated that the xpsZZZA region affects the activity of the phosphoglucomutase/phosphomannomutase enzyme, and the xpsZZZB region affects the mannoisomerase/phosphomannoisomerase activities. The xpsZV mutations affect the activity of the UDPG-pyrophosphorylase enzyme, and the xps VZ mutations affect the activity of the UDPG-dehydrogenase enzyme.