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

Colonies of strains carrying a stable lambda placMu15 translational fusion displayed sharply defined intense staining at the centre on Xgal medium. Th e fusion was in flu (ferric ion uptake), encoding an iron-regulated outer m embrane protein (IROMP) controlled via four overlapping ferric uptake regul ator (Fur) boxes in the sigma(70) promoter region. Fiu-LacZ was synthesized in low amounts (<1% of a transcriptional fiu::lacZ(+) fusion), localized t o membranes, and underwent processing from a large protein to one that co-m igrated with native beta-galactosidase, Intact cells synthesizing Fiu-LacZ often displayed greater enzymatic activity than permeabilized cells, The co lony centre was insensitive to iron regulation observed in liquid cultures and at the colony edge. Within colonies grown on 36 mu M iron citrate mediu m, fiu'-'lacZ protein fusion strains displayed 60-fold higher beta f-galact osidase activity in the centre, and transcriptional fiu::lacZ(+) fusion str ains displayed a 10-fold centre/edge difference. On medium without added ir on citrate, the centre/edge difference collapsed to <2.2-fold for both tran slational and transcriptional fusions because activity at the edge was dere pressed. Iran-insensitive fiu'-'lacZ expression in the colony centre occurr ed during a 6-18h time window at the start of colony morphogenesis, corresp onding to the initiation of multilayer microcolony development. A simple mo del for differential fiu'-'lacZ regulation is proposed whereby iron accessi bility changes during colony morphogenesis.