The role(s) in cell division of the Mycobacterium tuberculosis Rv0011c gene product, a homolog of the Streptomyces CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-M. tuberculosis CrgA (ECFP-CrgAMT) fusion protein is localized to the cell membrane, midcell, and cell pole regions in Mycobacterium smegmatis. Furthermore, the ECFP-CrgAMT fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in M. smegmatis. Bacterial two-hybrid assays indicated strong interactions of M. tuberculosis CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgAMT was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring. M. tuberculosis cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A M. smegmatis ΔcrgA strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a ΔcrgA strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.
On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.
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.
Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [14C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.
Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence of Neisseria meningitidis Z2491 revealed the presence of many repeats. One of these, designated REP2, contains a −24/−12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes are pilC1 and crgA, described as being involved in steps essential for the interaction of N. meningitidis with host cells. Furthermore, the REP2 sequences located upstream of pilC1 and crgA correspond to the previously identified promoters known to be induced during the initial localized adhesion of N. meningitidis with human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances as pilC1 and crgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion of N. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.
LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial–host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA–DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.
The crgA gene of Neisseria meningitidis, which codes for a LysR-type regulator, is divergently oriented with respect to the mdaB gene, which codes for a hypothetical NADPH-quinone oxidoreductase. Transcriptional studies of the intergenic region between crgA and mdaB showed that two overlapping and divergent promoters, PcrgA and PmdaB, control transcription of these genes. Deletion of the crgA gene led to a strong increase in transcription from the PcrgA promoter and a concomitant strong decrease in transcription from the PmdaB promoter, indicating that CrgA acts both as an autorepressor of transcription at its own promoter and as an activator of transcription at the mdaB promoter. Addition of α-methylene-γ-butyrolactone (MBL), an inducer of NADPH-quinone oxidoreductase, to wild-type N. meningitidis cells specifically resulted in further activation of transcription of the PmdaB promoter and more repression of transcription of the PcrgA promoter. No such regulation was observed when MBL was added to crgA-deficient cells, indicating that the transcriptional response to MBL is CrgA mediated. Under the same experimental conditions, no regulation of transcription by either CrgA or MBL was detected at the pilus and capsule genes. The role of CrgA in the regulation of gene expression during the infectious cycle of N. meningitidis is discussed.
The full length and the regulatory domain of the LysR-type transcriptional regulator CrgA have been crystallized. Diffraction data were collected from two crystal forms of full-length CrgA to 3.0 and 3.8 Å resolution, respectively. Crystals of the selenomethionine derivative of the C-terminal regulatory domain of CrgA diffracted to 2.3 Å resolution.
Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.2 M NDSB-256. Data were collected from two crystal forms of full-length CrgA belonging to space groups P212121 and P21, diffracting to 3.0 and 3.8 Å resolution and consistent with the presence of between six and ten and between ten and 20 copies of CrgA in the asymmetric unit, respectively. In addition, diffraction data were collected to 2.3 Å resolution from the selenomethionine derivative of the regulatory domain of CrgA. The crystals belonged to space group P21 and contained two molecules in the asymmetric unit.
CrgA; Neisseria meningitidis; LysR-type regulators
The conserved rodA and ftsW genes encode polytopic membrane proteins that are essential for bacterial cell elongation and division, respectively, and each gene is invariably linked with a cognate class B high-molecular-weight penicillin-binding protein (HMW PBP) gene. Filamentous differentiating Streptomyces coelicolor possesses four such gene pairs. Whereas rodA, although not its cognate HMW PBP gene, is essential in these bacteria, mutation of SCO5302 or SCO2607 (sfr) caused no gross changes to growth and septation. In contrast, disruption of either ftsW or the cognate ftsI gene blocked the formation of sporulation septa in aerial hyphae. The inability of spiral polymers of FtsZ to reorganize into rings in aerial hyphae of these mutants indicates an early pivotal role of an FtsW-FtsI complex in cell division. Concerted assembly of the complete divisome was unnecessary for Z-ring stabilization in aerial hyphae as ftsQ mutants were found to be blocked at a later stage in cell division, during septum closure. Complete cross wall formation occurred in vegetative hyphae in all three fts mutants, indicating that the typical bacterial divisome functions specifically during nonessential sporulation septation, providing a unique opportunity to interrogate the function and dependencies of individual components of the divisome in vivo.
Cryptococcal regulators of G protein signaling (CRG) are important for growth, differentiation, and virulence of Cryptococcus neoformans. Disruption of CRG2 resulted in dysregulated cAMP signaling and attenuated virulence, whereas disruption of CRG1 increased pheromone responses and enhanced virulence in the archetypal H99 strain. In tests with newly constructed near congenic mutants, a distinction between crg2Δ and crg1Δ gene expression was not apparent during macrophage interaction. Intranasal inoculation indicated that crg2Δ, crg1Δ, and wild-type strains reached the lungs within 0.5 hours of infection. However, CFUs were significantly decreased for crg2Δ at 2, 7, and 14 days post-infection. In contrast, crg1Δ proliferated to the same extent as the wild type (WT). Lung edema was not apparent in mice infected with crg2Δ 0.5 hours post-infection, which showed little cellular infiltrate in comparison to WT. Alveolar septal thickening was most evident in mice infected with crg1Δ, while mice infected with WT exhibited decreased septal thickening at later time points. Consistent with these observations, crg2Δ was less efficient in the elicitation of Th2 immune responses in a multiplex cytokine assay. Our results suggest that Crg2 is critical for establishment of early pulmonary infection and for persistence of infection, Crg1 regulates virulence in a strain-specific manner, and crg2Δ, crg1Δ and WT can all be distinguished on the basis of host tissue responses.
Cryptococcal regulators of G protein signaling; macrophage interaction; host response; cytokine production; and fungal virulence
We have characterized homologues of the bacterial cell division genes ftsL and divIC in the gram-positive mycelial bacterium Streptomyces coelicolor A3(2). We show by deletion-insertion mutations that ftsL and divIC are dispensable for growth and viability in S. coelicolor. When mutant strains were grown on a conventional rich medium (R2YE, containing high sucrose), inactivation of either ftsL or divIC resulted in the formation of aerial hyphae with partially constricted division sites but no clear separation of prespore compartments. Surprisingly, this phenotype was largely suppressed when strains were grown on minimal medium or sucrose-free R2YE, where division sites in many aerial hyphae had finished constricting and chains of spores were evident. Thus, osmolarity appears to affect the severity of the division defect. Furthermore, double mutant strains deleted for both ftsL and divIC are viable and have medium-dependent phenotypes similar to that of the single mutant strains, suggesting that functions performed by FtsL and DivIC are not absolutely required for septation during growth and sporulation. Alternatively, another division protein may partially compensate for the loss of both FtsL and DivIC on minimal medium or sucrose-free R2YE. Finally, based on transmission electron microscopy observations, we propose that FtsL and DivIC are involved in coordinating symmetrical annular ingrowth of the invaginating septum.
Prior to bacterial cell division, the ATP-dependent polymerization of the cytoskeletal protein, ParA, positions the newly replicated origin-proximal region of the chromosome by interacting with ParB complexes assembled on parS sites located close to the origin. During the formation of unigenomic spores from multi-genomic aerial hyphae compartments of Streptomyces coelicolor, ParA is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregation of tens of chromosomes into prespore compartments. Here, we show that in addition to being a segregation protein, ParA also interacts with the polarity protein, Scy, which is a component of the tip-organizing centre that controls tip growth. Scy recruits ParA to the hyphal tips and regulates ParA polymerization. These results are supported by the phenotype of a strain with a mutant form of ParA that uncouples ParA polymerization from Scy. We suggest that the ParA–Scy interaction coordinates the transition from hyphal elongation to sporulation.
chromosome segregation; ParA; polar growth; bacterial development; sporulation
In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.
We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.
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.
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.
The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.
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.
Differentiation; Development; Sporulation; Cell division; Chromosome segregation
The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans. A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MATα strains of the fungus. Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains. In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12α but not on the mitogen-activated protein kinase homolog Cpk1. The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1. Interestingly, crg1 mutants of MATα cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.
Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.
Chemical genetics uses small molecules to perturb biological systems to study gene function. By analogy with genetic lesions, chemical probes act as fast-acting, reversible, and “tunable” conditional alleles. Furthermore, small molecules can target multiple protein targets and target pathways simultaneously to uncover phenotypes that may be masked by genes encoding partially redundant proteins. Finally, potent chemical probes can be useful starting points for the development of human therapeutics. Here, we used cantharidin, a natural toxin, to uncover otherwise “hidden” phenotypes for a methyltransferase that has resisted characterization. This enzyme, Crg1, has no phenotype in standard conditions but is indispensible for survival in the presence of cantharidin. Using this chemical genetic relationship, we characterized novel functions of Crg1, and by combining diverse genomic assays with small molecule perturbation we characterized the mechanism of cantharidin cytotoxicity. These observations are relevant beyond yeast Crg1 because cantharidin and its analogues have potent anticancer activity, yet its therapeutic use has been limited to topical applications because of its cytotoxicity. Considering that methyltransferases are an extremely abundant and diverse class of cellular proteins, chemical probes such as cantharidin are critical for understanding their cellular roles and defining potential points of therapeutic intervention.
Ca2+ was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known. cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed in Streptomyces coelicolor M145. On R5− agar, the disruption of cabC resulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, while cabC overexpression significantly delayed development. Manipulation of the Ca2+ concentration in R5− agar could reverse the phenotypes of cabC disruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca2+ concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation in S. coelicolor. CabC most likely acts as a Ca2+ buffer and exerts its regulatory effects by controlling the intracellular Ca2+ concentration.
The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific ς factor encoded by whiG.
Members of the SMC (structural maintenance of chromosomes) protein family play a central role in higher-order chromosome dynamics from bacteria to humans. So far, studies of bacterial SMC proteins have focused only on unicellular rod-shaped organisms that divide by binary fission. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes. Here we focus on the contribution of SMC proteins to sporulation-associated chromosome segregation in S. coelicolor. Deletion of the smc gene causes aberrant DNA condensation and missegregation of chromosomes (7.5% anucleate spores). In vegetative mycelium, immunostained SMC proteins were observed sporadically, while in aerial hyphae about to undergo sporulation they appeared as irregularly spaced foci which accompanied but did not colocalize with ParB complexes. Our data demonstrate that efficient chromosome segregation requires the joint action of SMC and ParB proteins. SMC proteins, similarly to ParAB and FtsZ, presumably belong to a larger group of proteins whose expression is highly induced in response to the requirement of aerial hyphal maturation.
The identification of genes that predict in vitro cellular chemosensitivity of cancer cells is of great importance. Chemosensitivity related genes (CRGs) have been widely utilized to guide clinical and cancer chemotherapy decisions. In addition, CRGs potentially share functional characteristics and network features in protein interaction networks (PPIN).
In this study, we proposed a method to identify CRGs based on Gene Ontology (GO) and PPIN. Firstly, we documented 150 pairs of drug-CCRG (curated chemosensitivity related gene) from 492 published papers. Secondly, we characterized CCRGs from the perspective of GO and PPIN. Thirdly, we prioritized CRGs based on CCRGs’ GO and network characteristics. Lastly, we evaluated the performance of the proposed method.
We found that CCRG enriched GO terms were most often related to chemosensitivity and exhibited higher similarity scores compared to randomly selected genes. Moreover, CCRGs played key roles in maintaining the connectivity and controlling the information flow of PPINs. We then prioritized CRGs using CCRG enriched GO terms and CCRG network characteristics in order to obtain a database of predicted drug-CRGs that included 53 CRGs, 32 of which have been reported to affect susceptibility to drugs. Our proposed method identifies a greater number of drug-CCRGs, and drug-CCRGs are much more significantly enriched in predicted drug-CRGs, compared to a method based on the correlation of gene expression and drug activity. The mean area under ROC curve (AUC) for our method is 65.2%, whereas that for the traditional method is 55.2%.
Our method not only identifies CRGs with expression patterns strongly correlated with drug activity, but also identifies CRGs in which expression is weakly correlated with drug activity. This study provides the framework for the identification of signatures that predict in vitro cellular chemosensitivity and offers a valuable database for pharmacogenomics research.
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.