scholarly journals A Four-Dimensional View of Assembly of a Morphogenetic Protein during Sporulation in Bacillus subtilis

1999 ◽  
Vol 181 (3) ◽  
pp. 781-790 ◽  
Author(s):  
Kirsten D. Price ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Outer Surface ◽  
Mother Cell ◽  
Fluorescent Protein ◽  
Mature Spore ◽  
Time Lapse ◽  
Homologous Protein ◽  
C Terminus ◽  
Morphogenetic Protein ◽  
Green Fluorescent

ABSTRACT We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacteriumBacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, likespoIVA, are under the control of the mother cell transcription factor ςE. One such gene wasspoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.

scholarly journals Effects of Phosphorelay Perturbations on Architecture, Sporulation, and Spore Resistance in Biofilms of Bacillus subtilis

2006 ◽  
Vol 188 (8) ◽  
pp. 3099-3109 ◽  
Author(s):  
Jan-Willem Veening ◽  
Oscar P. Kuipers ◽  
Stanley Brul ◽  
Klaas J. Hellingwerf ◽  
Remco Kort
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Current Model ◽  
Laboratory Strain ◽  
Time Lapse ◽  
Rich Media ◽  
Flat Structures ◽  
Reporter Strains ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.

The “Pro” Sequence of the Sporulation-Specific ς Transcription Factor ςE Directs It to the Mother Cell Side of the Sporulation Septum

1999 ◽  
Vol 181 (19) ◽  
pp. 6171-6175 ◽  
Author(s):  
Jingliang Ju ◽  
W. G. Haldenwang
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Fluorescent Protein ◽  
Amino Terminus ◽  
Cell Compartment ◽  
Intracellular Location ◽  
Specific Protease ◽  
Green Fluorescent

ABSTRACT ςE, a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-ςE). Activation of ςE occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-ςE amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-ςE and green fluorescent protein (GFP) to visualize the intracellular location of pro-ςE by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-ςE::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-ςE::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-ςE::GFP, and presumably pro-ςE, to the mother cell is likely to be the reason why ςE activity is restricted to that compartment.

scholarly journals Heterochronic Phosphorelay Gene Expression as a Source of Heterogeneity in Bacillus subtilis Spore Formation

2010 ◽  
Vol 192 (8) ◽  
pp. 2053-2067 ◽  
Author(s):  
Imke G. de Jong ◽  
Jan-Willem Veening ◽  
Oscar P. Kuipers
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Large Cell ◽  
Time Lapse ◽  
Nutrient Sources ◽  
Bacillus Subtilis Spore ◽  
Artificial Induction ◽  
Limiting Nutrient ◽  
Green Fluorescent

ABSTRACT In response to limiting nutrient sources and cell density signals, Bacillus subtilis can differentiate and form highly resistant endospores. Initiation of spore development is governed by the master regulator Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay. Interestingly, only part of a clonal population will enter this developmental pathway, a phenomenon known as sporulation bistability or sporulation heterogeneity. How sporulation heterogeneity is established is largely unknown. To investigate the origins of sporulation heterogeneity, we constructed promoter-green fluorescent protein (GFP) fusions to the main phosphorelay genes and perturbed their expression levels. Using time-lapse fluorescence microscopy and flow cytometry, we showed that expression of the phosphorelay genes is distributed in a unimodal manner. However, single-cell trajectories revealed that phosphorelay gene expression is highly dynamic or “heterochronic” between individual cells and that stochasticity of phosphorelay gene transcription might be an important regulatory mechanism for sporulation heterogeneity. Furthermore, we showed that artificial induction or depletion of the phosphorelay phosphate flow results in loss of sporulation heterogeneity. Our data suggest that sporulation heterogeneity originates from highly dynamic and variable gene activity of the phosphorelay components, resulting in large cell-to-cell variability with regard to phosphate input into the system. These transcriptional and posttranslational differences in phosphorelay activity appear to be sufficient to generate a heterogeneous sporulation signal without the need of the positive-feedback loop established by the sigma factor SigH.

scholarly journals Assembly of an Oxalate Decarboxylase Produced under σK Control into the Bacillus subtilis Spore Coat

2004 ◽  
Vol 186 (5) ◽  
pp. 1462-1474 ◽  
Author(s):  
Teresa Costa ◽  
Leif Steil ◽  
Lígia O. Martins ◽  
Uwe Völker ◽  
Adriano O. Henriques
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Decarboxylase Activity ◽  
Spore Coat ◽  
Oxalate Decarboxylase ◽  
Bacillus Subtilis Spore ◽  
Morphogenetic Protein ◽  
Interior Layers ◽  
Coat Layer ◽  
Green Fluorescent

ABSTRACT Over 30 polypeptides are synthesized at various times during sporulation in Bacillus subtilis, and they are assembled at the surface of the developing spore to form a multilayer protein structure called the coat. The coat consists of three main layers, an amorphous undercoat close to the underlying spore cortex peptidoglycan, a lamellar inner layer, and an electron-dense striated outer layer. The product of the B. subtilis oxdD gene was previously shown to have oxalate decarboxylase activity when it was produced in Escherichia coli and to be a spore constituent. In this study, we found that OxdD specifically associates with the spore coat structure, and in this paper we describe regulation of its synthesis and assembly. We found that transcription of oxdD is induced during sporulation as a monocistronic unit under the control of σK and is negatively regulated by GerE. We also found that localization of a functional OxdD-green fluorescent protein (GFP) at the surface of the developing spore depends on the SafA morphogenetic protein, which localizes at the interface between the spore cortex and coat layers. OxdD-GFP localizes around the developing spore in a cotE mutant, which does not assemble the spore outer coat layer, but it does not persist in spores produced by the mutant. Together, the data suggest that OxdD-GFP is targeted to the interior layers of the coat. Additionally, we found that expression of a multicopy allele of oxdD resulted in production of spores with increased levels of OxdD that were able to degrade oxalate but were sensitive to lysozyme.

scholarly journals Polar Localization and Compartmentalization of ClpP Proteases during Growth and Sporulation in Bacillus subtilis

2008 ◽  
Vol 190 (20) ◽  
pp. 6749-6757 ◽  
Author(s):  
James Kain ◽  
Gina G. He ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Regulatory Networks ◽  
Fluorescent Protein ◽  
The Other ◽  
Spatial Control ◽  
Polar Localization ◽  
Cell Compartments ◽  
Green Fluorescent ◽  
Necessary And Sufficient

ABSTRACT Spatial control of proteolysis is emerging as a common feature of regulatory networks in bacteria. In the spore-forming bacterium Bacillus subtilis, the peptidase ClpP can associate with any of three ATPases: ClpC, ClpE, and ClpX. Here, we report that ClpCP, ClpEP, and ClpXP localize in foci often near the poles of growing cells and that ClpP and the ATPase are each capable of polar localization independently of the other component. A region of ClpC containing an AAA domain was necessary and sufficient for polar localization. We also report that ClpCP and ClpXP proteases differentially localize to the forespore and mother cell compartments of the sporangium during spore formation. Moreover, model substrates for each protease created by appending recognition sequences for ClpCP or ClpXP to the green fluorescent protein were preferentially eliminated from the forespore or the mother cell, respectively. Biased accumulation of ClpCP in the forespore may contribute to the cell-specific activation of the transcription factor σF by preferential ClpCP-dependent degradation of the anti-σF factor SpoIIAB.

scholarly journals Imaging and Analysis of Pseudomonas aeruginosa Swarming and Rhamnolipid Production

2011 ◽  
Vol 77 (23) ◽  
pp. 8310-8317 ◽  
Author(s):  
Joshua D. Morris ◽  
Jessica L. Hewitt ◽  
Lawrence G. Wolfe ◽  
Nachiket G. Kamatkar ◽  
Sarah M. Chapman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Nile Red ◽  
Temporal Distribution ◽  
Time Lapse ◽  
Content Type ◽  
Rhamnolipid Production ◽  
Serovar Typhimurium ◽  
Surface Motility ◽  
Green Fluorescent

ABSTRACTMany bacteria spread over surfaces by “swarming” in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacteriumPseudomonas aeruginosa. First, we quantify the temporal distribution ofP. aeruginosacells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming ofP. aeruginosaandSalmonella entericaserovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of severalP. aeruginosastrains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

scholarly journals A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

2004 ◽  
Vol 15 (10) ◽  
pp. 4622-4632 ◽  
Author(s):  
Yasmina Bauer ◽  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Hanspeter Helfer ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Hyphal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Ashbya Gossypii ◽  
Growth Guidance ◽  
Characteristic Features ◽  
Green Fluorescent ◽  
Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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