Differential fiu–lacZ fusion regulation linked to Escherichia coli colony development

Abstract

Colonies of strains carrying a stable λplacMu15 translational fusion displayed sharply defined intense staining at the centre on Xgal medium. The fusion was in fiu (ferric ion uptake), encoding an iron-regulated outer membrane protein (IROMP) controlled via four overlapping ferric uptake regulator (Fur) boxes in the σ70 promoter region. Fiu–LacZ was synthesized in low amounts (< 1% of a transcriptional fiu::lacZ+ fusion), localized to membranes, and underwent processing from a large protein to one that co-migrated with native β-galactosidase. Intact cells synthesizing Fiu–LacZ often displayed greater enzymatic activity than permeabilized cells. The colony centre was insensitive to iron regulation observed in liquid cultures and at the colony edge. Within colonies grown on 36 μM iron citrate medium, fiu'–'lacZ protein fusion strains displayed 60-fold higher β-galactosidase activity in the centre, and transcriptional fiu::lacZ+ fusion strains displayed a 10-fold centre/edge difference. On medium without added iron citrate, the centre/edge difference collapsed to < 2.2-fold for both translational and transcriptional fusions because activity at the edge was derepressed. Iron-insensitive fiu'–'lacZ expression in the colony centre occurred during a 6–18 h time window at the start of colony morphogenesis, corresponding to the initiation of multilayer microcolony development. A simple model for differential fiu'–'lacZ regulation is proposed whereby iron accessibility changes during colony morphogenesis.