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

Establishment and regulation of polar growth in Streptomyces

A fundamental question in developmental biology is how cells establish polarity, and most strikingly how cells grow polarly. From neuronal dendrites and root hairs to bud emergence and elongation of yeast, broadly conserved pathways control cell polarity in eukaryotes. In contrast, virtually nothing is known about the regulatory mechanisms controlling polar cell growth in prokaryotes. In evolutionary terms, the most ancient form of polar growth is found in the branching hyphae of the filamentous bacteria Streptomyces, and it is clear that the essential coiled-coil protein DivIVA, which forms part of a tip-organising, multiprotein polarisome complex, plays a key role in the control of cell polarity, apical growth and hyphal branching in Streptomyces coelicolor. I identified and characterised two regulatory mechanisms, both reminiscent of aspects of cell polarity control in eukaryotes. First, I show that the mechanistic basis of branch-site selection during hyphal growth in Streptomyces is a novel polarisome splitting mechanism, in which the apical tip polarisome splits to leave behind a small daughter polarisome on the lateral membrane as the tip grows away. This daughter polarisome gradually grows in size, and ultimately initiates the outgrowth of a new branch. Second, I show that the Ser/Thr protein kinase AfsK is part of an apparatus that controls the polarisome complex at the hyphal tip. Activated AfsK directly phosphorylates DivIVA and profoundly alters the subcellular localisation of DivIVA to establish multiple new sites of polar growth. Thereby, AfsK modulates apical growth and lateral branching during normal growth and cell wall stress. I suggest that this is part of a stress response that provides Streptomyces with a mechanism to dismantle the apical growth apparatus at established hyphal tips that encounter problems with cell wall synthesis (for example through exposure to an antibiotic or by hitting a physical obstacle in the soil) and instead direct emergence of new branches elsewhere along the hyphae

A flexible mathematical model platform for studying branching networks : experimentally validated using the model actinomycete, Streptomyces coelicolor

Branching networks are ubiquitous in nature and their growth often responds to environmental cues dynamically. Using the antibiotic-producing soil bacterium Streptomyces as a model we have developed a flexible mathematical model platform for the study of branched biological networks. Streptomyces form large aggregates in liquid culture that can impair industrial antibiotic fermentations. Understanding the features of these could aid improvement of such processes. The model requires relatively few experimental values for parameterisation, yet delivers realistic simulations of Streptomyces pellet and is able to predict features, such as the density of hyphae, the number of growing tips and the location of antibiotic production within a pellet in response to pellet size and external nutrient supply. The model is scalable and will find utility in a range of branched biological networks such as angiogenesis, plant root growth and fungal hyphal networks

Streptomyces Exploration is Triggered by Fungal Interactions and Volatile Signals

It has long been thought that the life cycle of Streptomyces bacteria encompasses three developmental stages: vegetative hyphae, aerial hyphae and spores. Here, we show interactions between Streptomyces and fungi trigger a previously unobserved mode of Streptomyces development. We term these Streptomyces cells ‘explorers’, for their ability to adopt a non-branching vegetative hyphal conformation and rapidly transverse solid surfaces. Fungi trigger Streptomyces exploratory growth in part by altering the composition of the growth medium, and Streptomyces explorer cells can communicate this exploratory behaviour to other physically separated streptomycetes using an airborne volatile organic compound (VOC). These results reveal that interkingdom interactions can trigger novel developmental behaviours in bacteria, here, causing Streptomyces to deviate from its classically-defined life cycle. Furthermore, this work provides evidence that VOCs can act as long-range communication signals capable of propagating microbial morphological switches

Contrôle spatial et temporel de la biosynthèse des acides mycoliques au cours du cycle cellulaire du pathogène résurgent Mycobacterium tuberculosis

La compréhension du mécanisme qui commande la coordination spatio-temporelle de la croissance et de la division de Mycobacterium tuberculosis, est essentielle pour lutter contre le bacille tuberculeux. La plupart des (nombreuses) enzymes de la synthèse du complexe acide mycolique-arabinogalactane-peptidoglycane de l'enveloppe sont essentielles à la survie du bacille. En utilisant une approche dynamique, nous avons localisé in vivo les enzymes du complexe Fatty-Acid-Synthase-II (FAS-II), impliquées dans la biosynthèse des acides mycoliques (AM), ainsi que leur transporteur Mmpl3. Les enzymes FAS-II co-localisent au niveau des pôles et du septa avec Wag31 : la protéine responsable de la polarisation de la biosynthèse du peptidoglycane mycobactérien. Mmpl3 se localise au niveau de l'enveloppe et se concentre dynamiquement aux pôles et aux septa. La localisation dynamique de FAS-II et du transporteur avec Wag31, au niveau des pôles de croissance et des septa, signifie que le composé principal de la mycomembrane pourrait être synthétisé à cet endroit précis. Ce constat met en évidence une différence majeure entre les mycobactéries et les autres bactéries en forme de bâtonnets étudiées à ce jour. Sur la base des activités polaires de biosynthèse de l'enveloppe déjà connus chez les mycobactéries, nous proposons l'existence d'une machinerie polaire complexe, consacrée à la biogenèse de l'ensemble de l'enveloppe. En conséquence, les pôles mycobactériens représenteraient le talon d'Achille du bacille à tous ses stades de croissance.Understanding the mechanism that controls the space-time coordination of the elongation and the cellular division of Mycobacterium tuberculosis is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of the cell wall Mycolic acid-Arabinogalactan-Peptidoglycan complex are essential for the bacterial life. Using a dynamic approach, we localized in vivo enzymes of the fatty acid synthase-II (FAS-II) complexes, involved in mycolic acid (MA) biosynthesis, together with their transporter MmpL3. The FAS-II enzymes co-locate at the poles and septa with Wag31: the protein responsible for the mycobacterial peptidoglycan biosynthesis polarization. MmpL3 was in the envelope, dynamically concentrated at the poles and septa. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and septa, suggests that the main components of the mycomembrane might be synthetized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known envelope biosynthetic polar activities in mycobacteria, we propose the existence of complex polar machines devoted to the biogenesis of the whole envelope. As a consequence, the mycobacterium pole would represent the Achilles' heel of the bacillus at all its growing stages

PopZ and FtsZ coordinate polar growth termination and cell division in Agrobacterium tumefaciens

Understanding how bacterial cells expand their cell walls is an important question with relevance to development of antibiotics. While many studies have focused on the regulation of bacterial elongation utilizing lateral cell wall biogenesis, polar growth in bacteria is less well understood. Yet, polar growth has been observed across taxonomically diverse bacteria like Actinobacteria and the alphaproteobacterial clade Rhizobiales (Howell and Brown, 2016). Interestingly, polar-growing bacteria within Rhizobiales lack canonical scaffolding proteins for spatial and temporal regulation of peptidoglycan synthesis during elongation. Here, we dissect the role of two candidate scaffolding proteins in directing cell wall synthesis in the bacterial plant pathogen, Agrobacterium tumefaciens. Since cell wall (peptidoglycan) biosynthesis during elongation and cell division is vital for bacterial survival, we expected many key proteins involved in these processes to be essential for cell survival. Thus, we developed a depletion system for A. tumefaciens (Figureroa-Cuilan et al. 2016). We further optimized a suite of target-specific fluorescent labeling techniques which allow us to visualize morphological changes during essential cell processes (Howell, Daniel, and Brown, 2017). We use these techniques to dissect the contributions of PopZ and FtsZ to polar growth and cell division. Although PopZ is not required for polar growth, it is required for proper coordination of polar growth, chromosome segregation, and cell division. This PopZ-mediated coordination ensures that daughter cells are the proper size and contain a complete complement of genetic material (Howell et al 2017). Next, we find that FtsZ is required for both termination of polar growth and cell division. This finding suggests that FtsZ has at least two important functions in regulation of cell wall biogenesis. First, FtsZ enables cell wall biogenesis machinery to be released or inactivated from the growth pole. Second, FtsZ must recruit additional proteins to mid cell to assemble the divisome, enabling activation of cell wall biogenesis to promote septum formation and cell separation. While further research is needed to understand how growth is targeted to the pole during elongation, our work provides mechanistic insights about the coordination of polar growth termination, chromosome segregation, and cell division. We hypothesize that our findings will be applicable to other closely related polar growing Rhizobiales, including plant, animal, and human pathogens.Includes bibliographical reference

ParH: a novel regulator of septum site placement in Streptomyces coelicolor

Streptomyces coelicolor is a Gram-positive, GC-rich, soil-dwelling, filamentous bacterium with a complex life cycle, which begins from a single uni-genomic spore. The life cycle is completed after the differentiation of multi-genomic aerial hyphae into uni-genomic spores. This process requires the segregation and organisation of many chromosomes of the sporogenic hyphae which are then compartmentalised by the synchronous placement of 20-50 septa, generating a single chromosome in each pre-spore compartment. Chromosome segregation and septum site-placement are two key components of cell division. Throughout the bacterial kingdom, these processes are controlled by the ParA/MinD superfamily of proteins. These proteins can be divided into two categories according to their function: those involved in chromosome segregation and those involved in septum-site placement. In S. coelicolor, the only characterised protein belonging to this superfamily is ParA that has been implicated in chromosome segregation along with its partner protein, ParB. Prior to septation, ParA forms long filaments along the length of the hyphae where they position ParB bound to the chromosomes. This study characterises a novel homologue of the ParA/MinD superfamily encoded by the gene SCO1772, which we have designated parH. We also characterised the gene (SCO1771), which is downstream of parH and translationally coupled. Through in vitro techniques such as analytical gel filtration, native-PAGE, chemical crosslinking, pelleting assays we have characterised their oligomerisation, determined their protein:protein interactions and present structural data for SCO1771. We show that ParH is involved in the determination of septum site placement during division. We also link ParH to both the chromosome segregation machinery and the tip organising centre (TIPOC), a multi protein assembly that drives growth. This work helps to clarify the link between chromosome segregation and septum site placement

Controlling growth and morphogenesis of the industrial enzyme producer Streptomyces lividans

Streptomyces are Gram-positive, soil dwelling bacteria that raised interest in the last 50 years for their high potential in antibiotic and protein production. Thanks to their saprophytic nature, streptomycetes secrete a massive amount of industrial enzymes. They have a relatively low level of endogenous extracellular proteolytic activity when compared to other expression hosts (e.g. Bacillus), they are generally more suited to produce proteins encoded by high G+C actinomycete genes in their native form, coupled to efficient secretion so as to avoid that the proteins end up in inclusion bodies (often a problem when using e.g. E. coli) and making downstream processes easier. Despite their attractive potential, Streptomyces present several constraints which so far limit their application in industry. The first constraint is morphology: by growing as a network of hyphae, they produce dense pellets in liquid cultures that hold Streptomyces back from being one of the first choice cell factories in large scale fermentations. In addition, the limited availability of efficient expression systems for high-level transcription/translation and subsequent secretion is a further bottleneck. This thesis presents the work done to address these issues for the optimization of Streptomyces lividans for future industrial applications and enzyme production.Microbial Biotechnolog

Investigation of Cell Division Genes in Streptomyces coelicolor

Streptomyces coelicolor is a Gram-positive, filamentous, high G-C content bacterium with a complex developmental life cycle involving differentiation into distinct tissues, such as the vegetative hyphae, aerial hyphae and spores. Unlike in other bacteria, cell division in Streptomyces. coelicolor is only required for sporulation rather than for viability. The key protein FtsZ, which assembles into Z-rings, marks the positions for future, regularly spaced septation that transforms the aerial hyphae into spores, is essential for septation during sporulation in S. coelicolor. The function of several genes, located between ftsZ (SCO2082) and divIVA (SCO2077) in the chromosome, have not been well characterised, despite the fact they are downstream of ftsZ in many Gram-positive bacteria, including Streptomyces. In this study we mainly focus on three genes SCO2081, SCO2080, SCO2079 (sepF), located downstream of ftsZ. SepF was previously shown to tether the Z-ring to the membrane in Bacillus. subtilis and promote FtsZ protofilament formation. Considering the chromosomal location, important roles in cell division or cell-wall synthesis were anticipated. In this work, we generated knockout mutant strains by the deletion of these three genes and confirmed the mutant strains generated. We characterized the mutant phenotypes using macroscopic observations and extensive microscopic analysis focusing on possible effects on the division process and cell-wall synthesis. We also monitored the localisation of the SepF protein during development of S. coelicolor in order to explore its role during the Z-ring assembly and positioning. The severe defect of septum formation in the the sepF (SCO2079) knockout mutant suggested a key role for SepF in the early stages of cell division in Streptomyces, which is different to the role of the B. subtilis SepF in the late stages of septum formation. The gene knockouts of the surrounding genes SCO2080 and SCO2081 resulted in less severe, more subtle phenotype, nevertheless affecting the efficiency of septation and cell division in Streptomyces

The streptomyces cytoskeletal protein (Scy) is a key component of the tip organising centre for polarized growth in streptomyces coelicolor

The Gram-positive bacterium Streptomyces coelicolor, is one of the main genetic model organisms in the phylum of the Actinobacteria. Streptomyces bacteria are soil dwelling filamentous bacteria with a complex life cycle consisting of multigenomic hyphae that then form unicellular spores. Bacterial cell shape determination has been influenced heavily by the discovery that bacteria have a number of eukaryotic cytoskeletal homologues as well as a number of accessory proteins unique to prokaryotes. As cell shape determination is dependent on the sites of insertion of new cell wall material, this is characteristically organised and driven by cytoskeletal proteins. Streptomyces coelicolor hyphal growth occurs through apical extension where new cell wall material is placed at the tips. This growth is driven in part by the cytoskeletal protein DivIVA. Here we characterise a novel Streptomyces cytoskeletal protein, Scy, encoded by the locus sco5397. Scy is a large protein with a novel coiled-coil 51-mer repeat structure. To study Scy, a scy knockout mutation was generated. The phenotype of the scy mutant suggests that it plays a significant role in cell shape, growth and chromosome positioning. Translational fluorescent protein fusions to scy were made and the subcellular localisation of Scy was determined to be strongly at growing hyphal tips. Further clarified here, Scy overexpression can recruit DivIVA protein and the cell wall synthesis machinery to new apical sites. The reciprocal is also shown whereby DivIVA overexpression can recruit Scy to new apical sites. Further to this in vivo and in vitro experiments were performed to determine that Scy and DivIVA interact, as well as the protein FilP encoded downstream of scy. The work here along with work in the field suggests that Scy forms part of a Tip Organising Centre (TIPOC) that alongside DivIVA, FilP, and numerous other proteins controls apical growth in the filamentous Streptomyces