Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response

The propensity of the bacterial rodlin protein RdlB to form amyloid fibrils determines its function in Streptomyces coelicolor.

Streptomyces bacteria form reproductive aerial hyphae that are covered with a pattern of pairwise aligned fibrils called rodlets. The presence of the rodlet layer requires two homologous rodlin proteins, RdlA and RdlB, and the functional amyloid chaplin proteins, ChpA-H. In contrast to the redundancy shared among the eight chaplins, both RdlA and RdlB are indispensable for the establishment of this rodlet structure. By using a comprehensive biophysical approach combined with in vivo characterization we found that RdlB, but not RdlA, readily assembles into amyloid fibrils. The marked difference in amyloid propensity between these highly similar proteins could be largely attributed to a difference in amino acid sequence at just three sites. Further, an engineered RdlA protein in which these three key amino acids were replaced with the corresponding residues from RdlB could compensate for loss of RdlB and restore formation of the surface-exposed amyloid layer in bacteria. Our data reveal that RdlB is a new functional amyloid and provide a biophysical basis for the functional differences between the two rodlin proteins. This study enhances our understanding of how rodlin proteins contribute to formation of an outer fibrillar layer during spore morphogenesis in streptomycetes

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants—which fail to properly localise FtsZ—could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades

Imaging of Streptomyces coelicolor A3(2) with Reduced Autofluorescence Reveals a Novel Stage of FtsZ Localization

Imaging of low abundance proteins in time and space by fluorescence microscopy is typically hampered by host-cell autofluorescence. Streptomycetes are an important model system for the study of bacterial development, and undergo multiple synchronous cell division during the sporulation stage. To analyse this phenomenon in detail, fluorescence microscopy, and in particular also the recently published novel live imaging techniques, require optimal signal to noise ratios. Here we describe the development of a novel derivative of Streptomyces coelicolor A3(2) with strongly reduced autofluorescence, allowing the imaging of fluorescently labelled proteins at significantly higher resolution. The enhanced image detail provided novel localization information for the cell division protein FtsZ, demonstrating a new developmental stage where multiple FtsZ foci accumulate at the septal plane. This suggests that multiple foci are sequentially produced, ultimately connecting to form the complete Z ring. The enhanced imaging properties are an important step forward for the confocal and live imaging of less abundant proteins and for the use of lower intensity fluorophores in streptomycetes

Bacterial size matters:Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections

Surge and swab pressure: A transient approach to running expandable assemblies

Shell is working on mono-diameter drilling (MOD). A MOD well, or MOD well section, has a (nearly) constant diameter over several sections. This is achieved by deploying expandable liners: a liner is lowered into position and is expanded by pulling an over-sized cone through it. This plasticly deforms the pipe, increasing its inner diameter to that of the previous section. MOD eliminates many of the constraints of conventional well design. Greater depths may be reached and zonal isolation may be performed without paying the penalty of reduced production rates.While lowering an expandable assembly (expandable liner and expansion tools) into a well, caution is required. Displacement of the drilling mud causes a pressure drop due to fluid friction and acceleration. The running speed of the assembly into the well is limited by the surge (overpressure) or swab (underpressure) pressure. Pressure must remain within a predefined window to ensure well control.The current understanding of the mechanisms governing surge/swab pressure, as well as the models used to predict it, are deemed insufficient. The effect of unorthodox MOD conditions is unknown. This study is conducted to overcome these obstacles.Firstly, a model is programmed so that transient surge and swab pressures, induced by assembly movement, may be predicted. The drilling mud’s velocity and pressure are described by the water hammer equations, which are solved using the interpolated method of characteristics. One dimensional (1D), unsteady and nonuniform flow of a slightly compressible fluid in a conduit with linearly elastic walls is considered. The annular pressure drop for flow with a moving inner pipe is solved considering both laminar and turbulent flow of a Herschel-Bulkley fluid. Flow over the expansion tools is solved separately and is considered 1D and incompressible.Secondly, the model is validated. The model is first validated under conventional conditions using field measurements presented in literature and performs better than existing models. The validity of the model while dealing with extremely small clearances, as found in MOD applications, is investigated through a full-scale experiment on Shell’s test rig. The surge and swab pressures are generally predictable. Some discrepancies between predictions and measurements are observed and are attributed to the relatively large uncertainty in clearance size and shape, which results from imperfect pipes. The surge/swab pressure’s sensitivity to the size and shape of the pipes means that predictions should be treated with care. The effect of local annular flow restriction over the expansion tools is investigated too. The tools are found to cause acceleration induced transient pressures not predicted by the model. This implies that the 1D and/or incompressible assumption at the interface does not describe the flow adequately.Thirdly, the effect of MOD well design at true well depths and assembly lengths is investigated through a case study. The surge pressure is significant. The pressure drop generated in the narrow annulus between assembly and wellbore is too high to allow significant flow. Instead, all drilling mud is displaced upwards through the drillstring. Swabbing occurs behind the assembly. Circulation while tripping in expandable assemblies is therefore impossible. At these depth and length scales, the effect of fluid compression and borehole expansion becomes significant, causing steady state models to become increasingly inaccurate with depth

SParticle, an algorithm for the analysis of filamentous, microorganisms in submerged cultures

Streptomycetes are filamentous bacteriathat produce a plethora of bioactive natural productsand industrial enzymes. Their mycelial lifestyle typicallyresults in high heterogeneity in bioreactors, withmorphologies ranging from fragments and openmycelial mats to dense pellets. There is a strongcorrelation between morphology and production insubmerged cultures, with small and open myceliafavouring enzyme production, while most antibioticsare produced mainly in pellets. Here we describeSParticle, a Streptomyces Particle analysis method thatcombines whole slide imaging with automated imageanalysis to characterize the morphology of submergedgrown Streptomyces cultures. SParticle allows theanalysis of over a thousand particles per hour, offeringa high throughput method for the imaging andstatistical analysis of mycelial morphologies. Thesoftware is available as a plugin for the open sourcesoftware ImageJ and allows users to create customfilters for other microbes. Therefore, SParticle is awidely applicable tool for the analysis of filamentousmicroorganisms in submerged culture

Autofluorescence of <i>S. coelicolor</i> M145 and FM145.

<p>Green autofluorescence (top two rows; 470–490 nm excitation and 515 long pass detection) and red autofluorescence (bottom two rows; 530–550 nm excitation and 590 long pass detection) in <i>S. coelicolor</i> FM145 (1^st column) and its parent M145 (3^rd column). Corresponding light images are presented in the 2^nd and 4^th columns, respectively. Exposure times are shown on the left side of the image. Autofluorescence is clearly reduced in FM145.</p