Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Abstract

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ϳ20 m h ؊1 , which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.Fluorescence microscopy has revolutionized our understanding of the bacterial cell and provided new opportunities to investigate the behavior of cell division proteins and chromosome dynamics in bacteria (25). Central to this research is the application of time-lapse microscopy to study bacterial cell division, which has revealed the complexity with which bacteria coordinate cellular growth and division. For example, the rodshaped bacteria Escherichia coli and Bacillus subtilis incorporate new peptidoglycan into their cell wall along their lateral walls, while coccoid bacteria such as Staphylococcus aureus do so at mid-cell (4). Actinobacteria, such as Corynebacterium and members of the mycelial, antibiotic-producing genus Streptomyces, incorporate peptidoglycan at the cell poles (4). In the case of streptomycetes, this allows them to adopt a hyphal growth strategy through peptidoglycan incorporation at the hyphal tip (9). This is ideally suited to the colonization of their particulate habitat, the soil, through the generation of a mycelium that permits nutrients to be transported from a nutrient reservoir to the actively growing tip. As such, streptomycetes represent a group of organisms that grow in a fashion distinct from other, better understood bacteria. The knowledge base associated with morphological and physiological differentiation in the model organism Streptomy...

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…Wonderful movies of septum formation during early growth of the hyphae of S. coelicolor show how irregular the placement of septa is in vegetative hyphae, with cross-walls dividing the vegetative hyphae into multigenomic compartments (276). In contrast, during sporulation-specific cell division in aerial hyphae, many septa are formed almost simultaneously and in a highly symmetrical fashion, followed by the formation of spore compartments and cell fission, resulting in chains of spores that each contain a single copy of the chromosome (reviewed in references 277 and 278).…”

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

“…To visualize the localization hierarchy of SsgB-eGFP and FtsZ-mCherry live during the initiation of sporulationspecific cell division, we further developed the technique of time-lapse imaging (Jyothikumar et al 2008) to image proteins in aerial hyphae. This unequivocally supported the fluorescence microscopy data, with SsgB localizing to cell division sites first, followed some minutes later by FtsZ ( Fig.…”

Replisome Trafficking in Growing Vegetative Hyphae ofStreptomyces coelicolorA3(2)

“…Moreover, replication is asynchronous, and only selected chromosomes undergo replication at any given time (17). Recently, a new method for monitoring Streptomyces hyphal growth in real time using time-lapse microscopy was established by Jyothikumar et al (11). It showed that, following germination, hyphal extension occurred at about 20 m h Ϫ1 when grown on mannitol-supplemented minimal medium at 30°C.…”