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.

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 centre. Mixed inocula of different strains show clonal exclusiveness as they proliferate outwards. 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. These observations indicate that bacterial growth on agar surfaces is a highly regulated process similar, in many respects, to the development of specific multicellular tissues and organisms.