Mechanistic Basis of Branch-Site Selection in Filamentous Bacteria

Many filamentous organisms, such as fungi, grow by tip-extension and by forming new branches behind the tips. A similar growth mode occurs in filamentous bacteria, including the genus Streptomyces, although here our mechanistic understanding has been very limited. The Streptomyces protein DivIVA is a critical determinant of hyphal growth and localizes in foci at hyphal tips and sites of future branch development. However, how such foci form was previously unknown. Here, we show experimentally that DivIVA focus-formation involves a novel mechanism in which new DivIVA foci break off from existing tip-foci, bypassing the need for initial nucleation or de novo branch-site selection. We develop a mathematical model for DivIVA-dependent growth and branching, involving DivIVA focus-formation by tip-focus splitting, focus growth, and the initiation of new branches at a critical focus size. We quantitatively fit our model to the experimentally-measured tip-to-branch and branch-to-branch length distributions. The model predicts a particular bimodal tip-to-branch distribution results from tip-focus splitting, a prediction we confirm experimentally. Our work provides mechanistic understanding of a novel mode of hyphal growth regulation that may be widely employed. Author Summary: Amongst the great variety of shapes that organisms assume, many grow in a filamentous manner and develop at least partly into a network of branches. Examples include plant roots, fungi and some bacteria. Whereas the mechanisms of filamentous growth are partially understood in fungi, the same cannot be said in filamentous bacteria, where our knowledge of hyphal growth regulation is very limited. To rectify this we have studied the bacteria Streptomyces, which are an excellent model for all hyphal bacteria. The protein DivIVA is known to play a critical role in controlling filamentous growth in Streptomyces, forming large foci at branch tips and smaller foci that mark sites of future branch outgrowth. However, until now nothing was known about how these foci first appear. We have shown experimentally that new foci appear via a novel mechanism, whereby existing tip-foci split into two clusters. The larger cluster remains at the growing tip, while the smaller cluster fixes onto the adjacent lateral membrane, where it grows in size, eventually initiating a new branch. By mathematically modelling how DivIVA foci grow, we show how this one simple mechanism of focus formation can quantitatively capture the statistical properties of the entire hyphal branching network.