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1.  Identification of new developmentally regulated genes involved in Streptomyces coelicolor sporulation 
BMC Microbiology  2013;13:281.
Background
The sporulation of aerial hyphae of Streptomyces coelicolor is a complex developmental process. Only a limited number of the genes involved in this intriguing morphological differentiation programme are known, including some key regulatory genes. The aim of this study was to expand our knowledge of the gene repertoire involved in S. coelicolor sporulation.
Results
We report a DNA microarray-based investigation of developmentally controlled gene expression in S. coelicolor. By comparing global transcription patterns of the wild-type parent and two mutants lacking key regulators of aerial hyphal sporulation, we found a total of 114 genes that had significantly different expression in at least one of the two mutants compared to the wild-type during sporulation. A whiA mutant showed the largest effects on gene expression, while only a few genes were specifically affected by whiH mutation. Seven new sporulation loci were investigated in more detail with respect to expression patterns and mutant phenotypes. These included SCO7449-7451 that affect spore pigment biogenesis; SCO1773-1774 that encode an L-alanine dehydrogenase and a regulator-like protein and are required for maturation of spores; SCO3857 that encodes a protein highly similar to a nosiheptide resistance regulator and affects spore maturation; and four additional loci (SCO4421, SCO4157, SCO0934, SCO1195) that show developmental regulation but no overt mutant phenotype. Furthermore, we describe a new promoter-probe vector that takes advantage of the red fluorescent protein mCherry as a reporter of cell type-specific promoter activity.
Conclusion
Aerial hyphal sporulation in S. coelicolor is a technically challenging process for global transcriptomic investigations since it occurs only as a small fraction of the colony biomass and is not highly synchronized. Here we show that by comparing a wild-type to mutants lacking regulators that are specifically affecting processes in aerial hypha, it is possible to identify previously unknown genes with important roles in sporulation. The transcriptomic data reported here should also serve as a basis for identification of further developmentally important genes in future functional studies.
doi:10.1186/1471-2180-13-281
PMCID: PMC3878966  PMID: 24308424
Differentiation; Aerial mycelium; Spore; Transcriptome; Spore pigment; Alanine dehydrogenase
2.  Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC–MS/MS 
Journal of Proteome Research  2013;12(9):4187-4192.
The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC–MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC–MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095.
doi:10.1021/pr400524d
PMCID: PMC3787806  PMID: 23905541
Ascore; DivIVA; phosphopeptides; phosphorylation site localization; ScaffoldPTM; targeted LC−MS/MS
3.  SIGNALS AND REGULATORS THAT GOVERN STREPTOMYCES DEVELOPMENT 
Fems Microbiology Reviews  2011;36(1):206-231.
Streptomyces coelicolor is the genetically best characterized species of a populous genus belonging to the Gram-positive Actinobacteria. Streptomycetes are filamentous soil organisms, well known for the production of a plethora of biologically active secondary metabolic compounds. The Streptomyces developmental life cycle is uniquely complex, and involves coordinated multicellular development with both physiological and morphological differentiation of several cell types, culminating in production of secondary metabolites and dispersal of mature spores. This review presents a current appreciation of the signaling mechanisms used to orchestrate the decision to undergo morphological differentiation, and the regulators and regulatory networks that direct the intriguing development of multigenomic hyphae, first to form specialized aerial hyphae, and then to convert them into chains of dormant spores. This current view of S. coelicolor development is destined for rapid evolution as data from “-omics” studies shed light on gene regulatory networks, new genetic screens identify hitherto unknown players, and the resolution of our insights into the underlying cell biological processes steadily improve.
doi:10.1111/j.1574-6976.2011.00317.x
PMCID: PMC3285474  PMID: 22092088
Differentiation; Development; Sporulation; Cell division; Chromosome segregation
4.  Mechanistic Basis of Branch-Site Selection in Filamentous Bacteria 
PLoS Computational Biology  2012;8(3):e1002423.
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.
doi:10.1371/journal.pcbi.1002423
PMCID: PMC3297577  PMID: 22423220
5.  The MreB-Like Protein Mbl of Streptomyces coelicolor A3(2) Depends on MreB for Proper Localization and Contributes to Spore Wall Synthesis▿ †  
Journal of Bacteriology  2011;193(7):1533-1542.
Most bacteria with a rod-shaped morphology contain an actin-like cytoskeleton consisting of MreB polymers, which form helical spirals underneath the cytoplasmic membrane to direct peptidoglycan synthesis for the elongation of the cell wall. In contrast, MreB of Streptomyces coelicolor is not required for vegetative growth but has a role in sporulation. Besides MreB, S. coelicolor encodes two further MreB-like proteins, Mbl and SCO6166, whose function is unknown. Whereas MreB and Mbl are highly similar, SCO6166 is shorter, lacking the subdomains IB and IIB of actin-like proteins. Here, we showed that MreB and Mbl are not functionally redundant but cooperate in spore wall synthesis. Expression analysis by semiquantitative reverse transcription-PCR revealed distinct expression patterns. mreB and mbl are induced predominantly during morphological differentiation. In contrast, sco6166 is strongly expressed during vegetative growth but switched off during sporulation. All genes could be deleted without affecting viability. Even a ΔmreB Δmbl double mutant was viable. Δsco6166 had a wild-type phenotype. ΔmreB, Δmbl, and ΔmreB Δmbl produced swollen, prematurely germinating spores that were sensitive to various kinds of stress, suggesting a defect in spore wall integrity. During aerial mycelium formation, an Mbl-mCherry fusion protein colocalized with an MreB-enhanced green fluorescent protein (MreB-eGFP) fusion protein at the sporulation septa. Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherry localization depended on the presence of a functional MreB protein. Our results revealed that MreB and Mbl cooperate in the synthesis of the thickened spore wall, while SCO6166 has a nonessential function during vegetative growth.
doi:10.1128/JB.01100-10
PMCID: PMC3067644  PMID: 21257777
6.  One of the Two Genes Encoding Nucleoid-Associated HU Proteins in Streptomyces coelicolor Is Developmentally Regulated and Specifically Involved in Spore Maturation▿ †  
Journal of Bacteriology  2009;191(21):6489-6500.
Streptomyces genomes encode two homologs of the nucleoid-associated HU proteins. One of them, here designated HupA, is of a conventional type similar to E. coli HUα and HUβ, while the other, HupS, is a two-domain protein. In addition to the N-terminal part that is similar to that of HU proteins, it has a C-terminal domain that is similar to the alanine- and lysine-rich C termini of eukaryotic linker histones. Such two-domain HU proteins are found only among Actinobacteria. In this phylum some organisms have only a single HU protein of the type with a C-terminal histone H1-like domain (e.g., Hlp in Mycobacterium smegmatis), while others have only a single conventional HU. Yet others, including the streptomycetes, produce both types of HU proteins. We show here that the two HU genes in Streptomyces coelicolor are differentially regulated and that hupS is specifically expressed during sporulation, while hupA is expressed in vegetative hyphae. The developmental upregulation of hupS occurred in sporogenic aerial hyphal compartments and was dependent on the developmental regulators whiA, whiG, and whiI. HupS was found to be nucleoid associated in spores, and a hupS deletion mutant had an average nucleoid size in spores larger than that in the parent strain. The mutant spores were also defective in heat resistance and spore pigmentation, although they possessed apparently normal spore walls and displayed no increased sensitivity to detergents. Overall, the results show that HupS is specifically involved in sporulation and may affect nucleoid architecture and protection in spores of S. coelicolor.
doi:10.1128/JB.00709-09
PMCID: PMC2795297  PMID: 19717607
7.  Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth in Streptomyces coelicolor▿ §  
Journal of Bacteriology  2008;190(22):7579-7583.
Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.
doi:10.1128/JB.00839-08
PMCID: PMC2576665  PMID: 18805980
8.  DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped Actinomycete Corynebacterium glutamicum▿ † 
Journal of Bacteriology  2008;190(9):3283-3292.
The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.
doi:10.1128/JB.01934-07
PMCID: PMC2347398  PMID: 18296522
9.  Influence of CrgA on Assembly of the Cell Division Protein FtsZ during Development of Streptomyces coelicolor 
Journal of Bacteriology  2006;188(4):1540-1550.
The product of the crgA gene of Streptomyces coelicolor represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In S. coelicolor, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.
doi:10.1128/JB.188.4.1540-1550.2006
PMCID: PMC1367258  PMID: 16452438
10.  Dynamics of FtsZ Assembly during Sporulation in Streptomyces coelicolor A3(2) 
Journal of Bacteriology  2005;187(9):3227-3237.
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.
doi:10.1128/JB.187.9.3227-3237.2005
PMCID: PMC1082811  PMID: 15838050
11.  Developmental Regulation of Transcription of whiE, a Locus Specifying the Polyketide Spore Pigment in Streptomyces coelicolor A3(2) 
Journal of Bacteriology  1998;180(9):2515-2521.
whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known “early” whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription from whiEP2. However, ςF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779–2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.
PMCID: PMC107196  PMID: 9573206

Results 1-11 (11)