The Use of Mudlac Transposons as Tools for Vital Staining to Visualize Clonal and Non-clonal Patterns of Organization in Bacterial Growth on Agar Surfaces

Abstract

When a histochemical stain for .beta.-galactosidase activity is applied to growth of gram-negative bacteria on agar medium, the pigmentation is non-uniform and capable of revealing internal colony organization into different cell types. Use of an Escherichia coli strain with a thermosensitive lac repressor indicates that colonies expand by addition of new cells at the periphery, and that older cells, which have synthesized .beta.-galactosidase early in development, remain in the center. Mixed inocula of different strains show clonal exclusiveness as they proliferate outwards. Phage Mudlac transposons can create genetic fusions that place .beta.-galactosidase expression under a variety of regulatory systems. Stained surface cultures of E. coli and Pseudomonas putida strains, carrying Mudlac insertions in plasmids, reveal a variety of flower-like staining patterns. These patterns display both clonal (i.e, sectorial) and non-clonal (circular and radial) features which are heritable within a given strain. The non-clonal aspects of the patterns reflect phenotypic differentiation without genetic change. Evidently, bacterial growth on agar surfaces is a highly regulated process similar to the development of specific multicellular tissues and organisms.