scholarly journals Genetic Interactions of smc, ftsK, and parB Genes in Streptomyces coelicolor and Their Developmental Genome Segregation Phenotypes

2008 ◽  
Vol 191 (1) ◽  
pp. 320-332 ◽  
Author(s):  
Rebekah M. Dedrick ◽  
Hans Wildschutte ◽  
Joseph R. McCormick
Keyword(s):  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Null Mutant ◽  
Triple Mutant ◽  
Developmentally Regulated ◽  
Spore Viability ◽  
Aerial Hyphae ◽  
Associated Proteins ◽  
Green Fluorescent ◽  
Insertion Mutants

ABSTRACT The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.

scholarly journals Developmental-Stage-Specific Assembly of ParB Complexes in Streptomyces coelicolor Hyphae

2005 ◽  
Vol 187 (10) ◽  
pp. 3572-3580 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Bertolt Gust ◽  
Jolanta Zakrzewska-Czerwinska ◽  
Keith F. Chater
Keyword(s):  
Green Fluorescent Protein ◽  
Developmental Stage ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Chromosome Partitioning ◽  
Aerial Hyphae ◽  
Green Fluorescent

ABSTRACT In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.

scholarly journals Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

2008 ◽  
Vol 74 (21) ◽  
pp. 6774-6781 ◽  
Author(s):  
Vinod Jyothikumar ◽  
Emma J. Tilley ◽  
Rashmi Wali ◽  
Paul R. Herron
Keyword(s):  
Protein Trafficking ◽  
Growth And Development ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Streptomyces Coelicolor ◽  
Time Lapse ◽  
Time Lapse Microscopy ◽  
Use Of Time ◽  
Aerial Hyphae ◽  
Green Fluorescent

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 ∼20 μm 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.

scholarly journals WhiD and WhiB, Homologous Proteins Required for Different Stages of Sporulation in Streptomyces coelicolor A3(2)

2000 ◽  
Vol 182 (5) ◽  
pp. 1286-1295 ◽  
Author(s):  
Virginie Molle ◽  
Wendy J. Palframan ◽  
Kim C. Findlay ◽  
Mark J. Buttner
Keyword(s):  
Cell Wall ◽  
Streptomyces Coelicolor ◽  
Mature Spore ◽  
Wall Thickening ◽  
Null Mutant ◽  
Chromosomal Dna ◽  
Homologous Proteins ◽  
Developmentally Regulated ◽  
Aerial Hyphae ◽  
Cell Wall Deposition

ABSTRACT The whiD locus, which is required for the differentiation of Streptomyces coelicolor aerial hyphae into mature spore chains, was localized by map-based cloning to the overlap between cosmids 6G4 and D63 of the minimal ordered library of Redenbach et al. (M. Redenbach et al., Mol. Microbiol. 21:77–96, 1996). Subcloning and sequencing showed that whiD encodes a homologue of WhiB, a protein required for the initiation of sporulation septation in S. coelicolor. WhiD and WhiB belong to a growing family of small (76- to 112-residue) proteins of unknown biochemical function in which four cysteines are absolutely conserved; all known members of this family are found in the actinomycetes. A constructed whiD null mutant showed reduced levels of sporulation, and those spores that did form were heat sensitive, lysed extensively, and were highly irregular in size, arising at least in part from irregularity in septum placement. The whiD null mutant showed extreme variation in spore cell wall deposition; most spores had uniformly thin (20- to 30-nm) walls, but spore chains were frequently observed in which there was irregular but very pronounced (up to 170 nm) cell wall thickening at the junctions between spores.whiD null mutant spores were frequently partitioned into irregular smaller units through the deposition of additional septa, which were often laid down in several different planes, very close to the spore poles. These “minicompartments” appeared to be devoid of chromosomal DNA. Two whiD promoters, whiDp1 andwhiDp2, were identified, and their activities were analyzed during development of wild-type S. coelicolor on solid medium. Both promoters were developmentally regulated;whiDp1 and whiDp2 transcripts were detected transiently, approximately at the time when sporulation septa were observed in the aerial hyphae.

scholarly journals Replisome Localization in Vegetative and Aerial Hyphae of Streptomyces coelicolor

2006 ◽  
Vol 188 (20) ◽  
pp. 7311-7316 ◽  
Author(s):  
Beata Ruban-Ośmiałowska ◽  
Dagmara Jakimowicz ◽  
Aleksandra Smulczyk-Krawczyszyn ◽  
Keith F. Chater ◽  
Jolanta Zakrzewska-Czerwińska
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Aerial Mycelium ◽  
Vegetative Mycelium ◽  
Single Compartment ◽  
Aerial Hyphae ◽  
Green Fluorescent ◽  
Replication Activity

ABSTRACT Using a functional fusion of DnaN to enhanced green fluorescent protein, we examined the subcellular localization of the replisome machinery in the vegetative mycelium and aerial mycelium of the multinucleoid organism Streptomyces coelicolor. Chromosome replication took place in many compartments of both types of hypha, with the apical compartments of the aerial mycelium exhibiting the highest replication activity. Within a single compartment, the number of “current” ongoing DNA replications was lower than the expected chromosome number, and the appearance of fluorescent foci was often heterogeneous, indicating that this process is asynchronous within compartments and that only selected chromosomes undergo replication.

scholarly journals Developmental Control of a parAB Promoter Leads to Formation of Sporulation-Associated ParB Complexes in Streptomyces coelicolor

2006 ◽  
Vol 188 (5) ◽  
pp. 1710-1720 ◽  
Author(s):  
Dagmara Jakimowicz ◽  
Sebastien Mouz ◽  
Jolanta Zakrzewska-Czerwińska ◽  
Keith F. Chater
Keyword(s):  
Fluorescent Protein ◽  
Developmental Regulation ◽  
Streptomyces Coelicolor ◽  
Proximal Part ◽  
Complex Life Cycle ◽  
Aerial Hyphae ◽  
Specific Induction ◽  
Dna Partitioning ◽  
Green Fluorescent ◽  
Promoter Mutations

ABSTRACT The Streptomyces coelicolor partitioning protein ParB binds to numerous parS sites in the oriC-proximal part of the linear chromosome. ParB binding results in the formation of large complexes, which behave differentially during the complex life cycle (D. Jakimowicz, B. Gust, J. Zakrzewska-Czerwinska, and K. F. Chater, J. Bacteriol. 187:3572-3580, 2005). Here we have analyzed the transcriptional regulation that underpins this developmentally specific behavior. Analysis of promoter mutations showed that the irregularly spaced complexes present in vegetative hyphae are dependent on the constitutive parABp 1 promoter, while sporulation-specific induction of the promoter parABp 2 is required for the assembly of arrays of ParB complexes in aerial hyphae and thus is necessary for efficient chromosome segregation. Expression from parABp 2 depended absolutely on two sporulation regulatory genes, whiA and whiB, and partially on two others, whiH and whiI, all four of which are needed for sporulation septation. Because of this pattern of dependence, we investigated the transcription of these four whi genes in whiA and whiB mutants, revealing significant regulatory interplay between whiA and whiB. A strain in which sporulation septation (but not vegetative septation) was blocked by mutation of a sporulation-specific promoter of ftsZ showed close to wild-type induction of parABp 2 and formed fairly regular ParB-enhanced green fluorescent protein foci in aerial hyphae, ruling out strong morphological coupling or checkpoint regulation between septation and DNA partitioning during sporulation. A model for developmental regulation of parABp 2 expression is presented.

scholarly journals Dynamics of FtsZ Assembly during Sporulation in Streptomyces coelicolor A3(2)

2005 ◽  
Vol 187 (9) ◽  
pp. 3227-3237 ◽  
Author(s):  
Nina Grantcharova ◽  
Ulrika Lustig ◽  
Klas Flärdh
Keyword(s):  
Time Course ◽  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Hyphal Cell ◽  
Ring Assembly ◽  
Aerial Hyphae ◽  
Z Ring ◽  
Ftsz Assembly ◽  
Green Fluorescent

ABSTRACT FtsZ, the bacterial tubulin homologue, is the main player in at least two distinct processes of cell division during the development of Streptomyces coelicolor A3(2). It forms cytokinetic rings and is required for the formation of both the widely spaced hyphal cross walls in the substrate mycelium and the specialized septation that converts sporogenic aerial hyphae into spores. The latter developmentally controlled septation involves the coordinated assembly of large numbers of FtsZ rings in each sporulating hyphal cell. We used an FtsZ-enhanced green fluorescent protein (EGFP) translational fusion to visualize the progression of FtsZ ring assembly in vivo during sporulation of aerial hyphae. This revealed that the regular placement of multiple FtsZ rings and initiation of cytokinesis was preceded by a protracted phase during which spiral-shaped FtsZ intermediates were detected along the length of the aerial hyphal cell. Time course experiments indicated that they were remodeled and gradually replaced by regularly spaced FtsZ rings. Such spiral-shaped filaments could also be detected with immunofluorescence microscopy using an antiserum against FtsZ. Based on our observations, we propose a model for the progression of Z-ring assembly during sporulation of S. coelicolor. Furthermore, mutants lacking the developmental regulatory genes whiA, whiB, whiG, whiH, and whiI were investigated. They failed in up-regulation of the expression of FtsZ-EGFP in aerial hyphae, which is consistent with the known effects of these genes on ftsZ transcription.

scholarly journals The PWWP Domain of Dnmt3a and Dnmt3b Is Required for Directing DNA Methylation to the Major Satellite Repeats at Pericentric Heterochromatin

2004 ◽  
Vol 24 (20) ◽  
pp. 9048-9058 ◽  
Author(s):  
Taiping Chen ◽  
Naomi Tsujimoto ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Regulatory Region ◽  
Pericentric Heterochromatin ◽  
Pwwp Domain ◽  
Major Satellite ◽  
Satellite Repeats ◽  
Associated Proteins ◽  
Green Fluorescent

ABSTRACT Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.

scholarly journals Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

2006 ◽  
Vol 5 (8) ◽  
pp. 1328-1336 ◽  
Author(s):  
Takashi Kikuma ◽  
Mamoru Ohneda ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Conidial Germination ◽  
Aerial Hyphae ◽  
Green Fluorescent ◽  
Starvation Conditions

ABSTRACT Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.

scholarly journals MafB Is Essential for Renal Development and F4/80 Expression in Macrophages

2006 ◽  
Vol 26 (15) ◽  
pp. 5715-5727 ◽  
Author(s):  
Takashi Moriguchi ◽  
Michito Hamada ◽  
Naoki Morito ◽  
Tsumoru Terunuma ◽  
Kazuteru Hasegawa ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Fetal Liver ◽  
Primary Cultures ◽  
Hematopoietic Cells ◽  
Null Mutant ◽  
Macrophage Population ◽  
Activation Domains ◽  
Null Mutant Mice ◽  
Green Fluorescent

ABSTRACT MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. mafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

scholarly journals Reciprocal Regulation between SigK and Differentiation Programs in Streptomyces coelicolor

2009 ◽  
Vol 191 (21) ◽  
pp. 6473-6481 ◽  
Author(s):  
Xu-Ming Mao ◽  
Zhan Zhou ◽  
Xiao-Ping Hou ◽  
Wen-Jun Guan ◽  
Yong-Quan Li
Keyword(s):  
Fluorescent Protein ◽  
Streptomyces Coelicolor ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Reciprocal Regulation ◽  
Enhanced Production ◽  
In The Wild ◽  
Negative Role ◽  
Green Fluorescent ◽  
Similar Dynamic

ABSTRACT Here we reported that deletion of SigK (SCO6520), a sigma factor in Streptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression of chpE and chpH than that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression of actII-orf4 and redD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, the sigK promoter (sigKp) activity gradually increased and sigK expression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoter ermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed under sigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed under ermEp*. These data suggested that the climbing expression of sigK might reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.

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