RESUMEN
The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.
Asunto(s)
Pared Celular/química , Streptomyces coelicolor/citología , Ácidos Teicoicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Pared Celular/metabolismo , Microscopía por Crioelectrón , Regulación Bacteriana de la Expresión Génica , Peptidoglicano/química , Peptidoglicano/metabolismo , Polisacáridos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrollo , Ácidos Teicoicos/química , Tomografía/métodosRESUMEN
Reconciling transcription and DNA replication in the growing hyphae of the filamentous bacterium Streptomyces presents several physical constraints on growth due to their apically extending and branching, multigenomic cells and chromosome replication being independent of cell division. Using a GFP translational fusion to the ß'-subunit of RNA polymerase (rpoC-egfp), in its native chromosomal location, we observed growing Streptomyces hyphae using time-lapse microscopy throughout the lifecycle and under different growth conditions. The RpoC-eGFP fusion co-localized with DNA around 1.8 µm behind the extending tip, whereas replisomes localize around 4-5 µm behind the tip, indicating that at the growing tip, transcription and chromosome replication are to some degree spatially separated. Dual-labelled RpoC-egfp/DnaN-mCherry strains also indicate that there is limited co-localization of transcription and chromosome replication at the extending hyphal tip. This likely facilitates the use of the same DNA molecule for active transcription and chromosome replication in growing cells, independent of cell division. This represents a novel, but hitherto unknown mechanism for reconciling two fundamental processes that utilize the same macromolecular template that allows for rapid growth without compromising chromosome replication in filamentous bacteria and may have implications for evolution of filamentous growth in micro-organisms, where uncoupling of DNA replication from cell division is required.
Asunto(s)
Replicación del ADN , Streptomyces coelicolor/genética , Transcripción Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Ligasas/genética , Microscopía por Video , Nitrógeno/deficiencia , Biosíntesis de Proteínas , Streptomyces coelicolor/citología , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
otrA resembles elongation factor G (EF-G) and is considered to be an oxytetracycline (OTC)-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE* promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coelicolor M145 by intergeneric conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M145-OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin (Act) production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that the transcriptional level of pathway-specific regulatory gene actII-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Streptomyces coelicolor/efectos de los fármacos , Antraquinonas/metabolismo , Farmacorresistencia Bacteriana/genética , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismoRESUMEN
Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular/genética , Eliminación de Gen , Esporas Bacterianas/fisiología , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Peptidoglicano/biosíntesis , Filogenia , Transporte de Proteínas , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/fisiologíaRESUMEN
Growth of Streptomyces in submerged culture is characterized by the formation of complex mycelial particles, known as pellets or clumps, which strongly influence antibiotic production. Also, many bioactive molecules produced by Streptomyces have great potential to modulate soil bacteria morphological development. However, there has been no effort directed at engineering mycelial morphology using these small molecules. Here, thiostrepton was identified, using a combination of qRT-PCR, semi-preparative HPLC, and MALDI-TOF MS, as a pellet-inducing compound produced by S. laurentii ATCC31255. At sub-inhibitory concentration, thiostrepton stimulated Streptomyces coelicolor M145 pellet formation and antibiotics production were altered, with 3-fold and 2-fold decreases in actinorhodin and undecylprodigiosin yields, respectively. It was also shown that mycelial morphology can be influenced by other antibiotic class at sub-inhibitory concentrations. For instance, in the presence of spectinomycin, S. coelicolor M145, which under typical growth conditions forms large diameter pellets with many protruding hyphae, instead formed small diameter pellets with barely visible hyphae at the edge. Importantly, this morphology produced a 4-fold increase in undecylprodigiosin production and 3-fold decrease in actinorhodin production. These results indicated that these small molecules, previously identified as antimicrobials, also have great potential for influencing mycelial morphology.
Asunto(s)
Antibacterianos/farmacología , Streptomyces coelicolor/citología , Streptomyces coelicolor/efectos de los fármacos , Streptomyces/metabolismo , Tioestreptona/farmacología , Antibacterianos/biosíntesis , Medios de Cultivo , Pruebas de Sensibilidad Microbiana , Micelio/citología , Micelio/efectos de los fármacos , Micelio/crecimiento & desarrollo , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrollo , Tioestreptona/biosíntesis , Tioestreptona/aislamiento & purificaciónRESUMEN
Cell wall glycopolymers (CWG) represent an important component of the Gram-positive cell envelope with many biological functions. The mycelial soil bacterium Streptomyces coelicolor A3(2) incorporates two distinct CWGs, polydiglycosylphosphate (PDP) and teichulosonic acid, into the cell wall of its vegetative mycelium but only little is known about their role in the complex life cycle of this microorganism. In this study we established assays to measure the total amount of CWGs in mycelial cell walls and spore walls, to quantify the individual CWGs and to determine the length of PDP. By applying these assays, we discovered that the relative amount of CWGs, especially of PDP, is reduced in spores compared to vegetative mycelium. Furthermore we found that PDP extracted from mycelial cell walls consisted of at least 19 repeating units, whereas spore walls contained substantially longer PDP polymers.
Asunto(s)
Pared Celular/química , Streptomyces coelicolor/química , Colorimetría , Galactosa/química , Hexosaminas/química , Esporas Bacterianas/química , Streptomyces coelicolor/citología , Ácidos Urónicos/químicaRESUMEN
In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.
Asunto(s)
Carbono/metabolismo , Metabolismo Secundario , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Antibacterianos/farmacología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Glucosa/farmacología , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Metabolismo Secundario/efectos de los fármacos , Metabolismo Secundario/genética , Streptomyces coelicolor/efectos de los fármacos , Streptomyces coelicolor/genéticaRESUMEN
The molecular mechanisms regulating tryptophan biosynthesis in actinomycetes are poorly understood; similarly, the possible roles of tryptophan in the differentiation program of microorganism life-cycle are still underexplored. To unveil the possible regulatory effect of this amino acid on gene expression, an integrated study based on quantitative teverse transcription-PCR (qRT-PCR) and proteomic approaches was performed on the actinomycete model Streptomyces coelicolor. Comparative analyses on the microorganism growth in a minimal medium with or without tryptophan supplementation showed that biosynthetic trp gene expression in S. coelicolor is not subjected to a negative regulation by the presence of the end product. Conversely, tryptophan specifically induces the transcription of trp genes present in the biosynthetic gene cluster of the calcium-dependent antibiotic (CDA), a lipopeptide containing D- and L-tryptophan residues. In addition, tryptophan stimulates the transcription of the CDA gene cluster regulator cdaR and, coherently, CDA production. Surprisingly, tryptophan also promotes the production of actinorhodin, another antibiotic that does not contain this amino acid in its structure. Combined 2D-DIGE and nano liquid chromatography electrospray linear ion trap tandem mass spectrometry (LC-ESI-LIT-MS/MS) analyses revealed that tryptophan exerts a growth-stage-dependent global effect on S. coelicolor proteome, stimulating anabolic pathways and promoting the accumulation of key factors associated with morphological and physiological differentiation at the late growth stages. Phenotypic observations by scanning electron microscopy and spore production assays demonstrated an increased sporulation in the presence of tryptophan. Transcriptional analysis of catabolic genes kynA and kynB suggested that the actinomycete also uses tryptophan as a carbon and nitrogen source. In conclusion, this study originally provides the molecular basis underlying the stimulatory effect of tryptophan on the production of antibiotics and morphological development program of this actinomycete.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Streptomyces coelicolor/citología , Streptomyces coelicolor/fisiología , Triptófano/metabolismo , Carbono/metabolismo , Cromatografía Liquida , Medios de Cultivo/química , Electroforesis en Gel Bidimensional , Metabolismo Energético , Perfilación de la Expresión Génica , Microscopía Electrónica de Rastreo , Nitrógeno/metabolismo , Proteoma/análisis , Espectrometría de Masa por Ionización de Electrospray , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/ultraestructura , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismoRESUMEN
During morphological differentiation of Streptomyces coelicolor A3(2), the sporogenic aerial hyphae are transformed into a chain of more than fifty spores in a highly coordinated manner. Synthesis of the thickened spore envelope is directed by the Streptomyces spore wall synthesizing complex SSSC which resembles the elongasome of rod-shaped bacteria. The SSSC includes the eukaryotic type serine/threonine protein kinase (eSTPK) PkaI, encoded within a cluster of five independently transcribed eSTPK genes (SCO4775-4779). To understand the role of PkaI in spore wall synthesis, we screened a S. coelicolor genomic library for PkaI interaction partners by bacterial two-hybrid analyses and identified several proteins with a documented role in sporulation. We inactivated pkaI and deleted the complete SCO4775-4779 cluster. Deletion of pkaI alone delayed sporulation and produced some aberrant spores. The five-fold mutant NLΔ4775-4779 had a more severe defect and produced 18% aberrant spores affected in the integrity of the spore envelope. Moreover, overbalancing phosphorylation activity by expressing a second copy of any of these kinases caused a similar defect. Following co-expression of pkaI with either mreC or pbp2 in E. coli, phosphorylation of MreC and PBP2 was demonstrated and multiple phosphosites were identified by LC-MS/MS. Our data suggest that elaborate protein phosphorylation controls activity of the SSSC to ensure proper sporulation by suppressing premature cross-wall synthesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión a las Penicilinas/metabolismo , Fosforilación , Unión Proteica , Esporas Bacterianas/metabolismo , Espectrometría de Masas en TándemRESUMEN
The angucycline antibiotic jadomycin B (JdB) produced by Streptomyces venezuelae has been found here to induce complex survival responses in Streptomyces coelicolor at subinhibitory concentration. The receptor for JdB was identified as a "pseudo" gamma-butyrolactone receptor, ScbR2, which was shown to bind two previously unidentified target promoters, those of redD (redDp) and adpA (adpAp), thus directly regulating undecylprodigiosin (Red) production and morphological differentiation, respectively. Because AdpA also directly regulates the expression of redD, ScbR2, AdpA, and RedD together form a feed-forward loop controlling both differentiation and Red production phenotypes. Different signal strengths (i.e., JdB concentrations) were shown to induce the two different phenotypes by modulating the relative transcription levels of adpA vs. redD. The induction of morphological differentiation and endogenous antibiotic production by exogenous antibiotic exemplifies an important survival strategy more sophisticated than the induction of antibiotic resistance.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Isoquinolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/fisiología , Streptomyces coelicolor/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Mediciones Luminiscentes , Regiones Promotoras Genéticas/genética , Streptomyces coelicolor/citología , Streptomyces coelicolor/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by SCO4598, SCO4597, and SCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in Streptomyces coelicolor and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene, abrC3 (SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of abrC3 in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the ΔabrC3 mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the actII-ORF4 promoter region; this was independently verified by in vitro DNA-binding assays. This binding is dependent on the sequence 5'-GAASGSGRMS-3'. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the redZ or redD promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of SCO0736, bdtA (SCO3328), absR1 (SCO6992), and SCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ΔabrC3 mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.
Asunto(s)
Antibacterianos/biosíntesis , Regulación Bacteriana de la Expresión Génica , Regulón , Streptomyces coelicolor/genética , Factores de Transcripción/metabolismo , Antraquinonas/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , ADN Bacteriano/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Unión Proteica , Streptomyces coelicolor/citologíaRESUMEN
Streptomycetes are filamentous soil bacteria that are used in industry for the production of enzymes and antibiotics. When grown in bioreactors, these organisms form networks of interconnected hyphae, known as pellets, which are heterogeneous in size. Here we describe a method to analyze and sort mycelial pellets using a Complex Object Parametric Analyzer and Sorter (COPAS). Detailed instructions are given for the use of the instrument and the basic statistical analysis of the data. We furthermore describe how pellets can be sorted according to user-defined settings, which enables downstream processing such as the analysis of the RNA or protein content. Using this methodology the mechanism underlying heterogeneous growth can be tackled. This will be instrumental for improving streptomycetes as a cell factory, considering the fact that productivity correlates with pellet size.
Asunto(s)
Citometría de Flujo/métodos , Streptomyces coelicolor/citología , Citometría de Flujo/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
In Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation. Furthermore, overexpression of redD or actII-orf4 in the proteasome-deficiency mutant resulted in SigT degradation and over-production of both undecylprodigiosin and actinorhodin. Therefore the proteasome is required for SigT degradation by affecting the production of secondary metabolites during cell differentiation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Diferenciación Celular , Complejo de la Endopetidasa Proteasomal/metabolismo , Factor sigma/metabolismo , Streptomyces coelicolor/citología , Streptomyces coelicolor/metabolismo , Antraquinonas/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , ProteolisisRESUMEN
Streptomycetes are mycelium-forming bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in industrial bioreactors tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this work the relationship between differentiation and antibiotic production in lab-scale bioreactors was defined. Streptomyces coelicolor was used as a model strain. Morphological differentiation was comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation was demonstrated to be one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in bioreactors.
Asunto(s)
Apoptosis , Reactores Biológicos/microbiología , Micelio/citología , Micelio/crecimiento & desarrollo , Prodigiosina/análogos & derivados , Streptomyces coelicolor/crecimiento & desarrollo , Antraquinonas/metabolismo , Antibacterianos/biosíntesis , Técnicas de Cultivo Celular por Lotes , Fermentación , Prodigiosina/biosíntesis , Streptomyces coelicolor/citología , Factores de TiempoRESUMEN
The whiH gene is required for the orderly sporulation septation that divides aerial hyphae into spores in Streptomyces coelicolor. Here, we use a whiHp-mCherry transcriptional reporter construct to show that whiHp is active specifically in aerial hyphae, fluorescence being dependent on sporulation sigma factor WhiG. The results show that the promoter is active before the septation event that separates the subapical compartment from the tip compartment destined to become a spore chain. We conclude that WhiG-directed RNA polymerase activity, which is required for whiH transcription, must precede this septation event and is not restricted to apical sporogenic compartment of the aerial hyphae. Further, it is demonstrated that WhiH, a predicted member of the GntR family of transcription factors, is able to bind specifically to a sequence in its own promoter, strongly suggesting that it acts as an autoregulatory transcription factor. Finally, we show by site-directed mutagenesis and a genetic complementation test that whiH is translated from a start codon overlapping with the previously identified transcription start point, implying leaderless transcription.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Esporas Bacterianas/crecimiento & desarrollo , Streptomyces coelicolor/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hifa/genética , Hifa/crecimiento & desarrollo , Hifa/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Factor sigma/genética , Factor sigma/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas de Transporte de Membrana/metabolismo , Transporte de Proteínas/fisiología , Streptomyces coelicolor/metabolismo , Proteínas Bacterianas/genética , Escherichia coli , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Plásmidos , Proteínas Recombinantes , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Imagen de Lapso de TiempoRESUMEN
The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.
Asunto(s)
ADP Ribosa Transferasas/deficiencia , Antibacterianos/biosíntesis , Eliminación de Gen , Streptomyces coelicolor/enzimología , Medios de Cultivo/química , Presión Osmótica , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Esporas Bacterianas/citología , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismoRESUMEN
Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes.
Asunto(s)
GMP Cíclico/análogos & derivados , Hidrolasas Diéster Fosfóricas/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/metabolismo , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Mutación , Hidrolasas Diéster Fosfóricas/genética , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Streptomyces coelicolor/citología , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
Successful application of a computational model for rational design of industrial Streptomyces exploitation requires a better understanding of the relationship between morphology-dictated by microbial growth, branching, fragmentation and adhesion-and product formation. Here we review the state-of-the-art in modeling of growth and product formation by filamentous microorganisms and expand on existing models by combining a morphological and structural approach to realistically model and visualize a three-dimensional pellet. The objective is to provide a framework to study the effect of morphology and structure on natural product and enzyme formation and yield. Growth and development of the pellet occur via the processes of apical extension, branching and cross-wall formation. Oxygen is taken to be the limiting component, with the oxygen concentration at the tips regulating growth kinetics and the oxygen profile within the pellet affecting the probability of branching. Biological information regarding the processes of differentiation and branching in liquid cultures of the model organism Streptomyces coelicolor has been implemented. The model can be extended based on information gained in fermentation trials for different production strains, with the aim to provide a test drive for the fermentation process and to pre-assess the effect of different variables on productivity. This should aid in improving Streptomyces as a production platform in industrial biotechnology.
Asunto(s)
Productos Biológicos/metabolismo , Biotecnología/métodos , Microbiología Industrial/métodos , Streptomyces coelicolor/citología , Streptomyces coelicolor/genética , Reactores Biológicos/microbiología , Simulación por Computador , Fermentación , Oxígeno/metabolismo , Streptomyces coelicolor/crecimiento & desarrollo , Streptomyces coelicolor/metabolismoRESUMEN
Morphology is important in industrial processes involving filamentous organisms because it affects the mixing and mass transfer and can be linked to productivity. Image analysis provides detailed information about the morphology but, in practice, it is often laborious including both collection of high quality images and image processing. Laser diffraction is rapid and fully automatic and provides a volume-weighted distribution of the particle sizes. However, it is based on a number of assumptions that do not always apply to samples. We have evaluated laser diffraction to measure cell clumps and pellets of Streptomyces coelicolor compare to image analysis. Samples, taken five times during fed-batch cultivation, were analyzed by image analysis and laser diffraction. The volume-weighted size distribution was calculated for each sample. Laser diffraction and image analysis yielded similar size distributions, i.e. unimodal or bimodal distributions. Both techniques produced similar estimations of the population means, whereas the estimates of the standard deviations were generally higher using laser diffraction compared to image analysis. Therefore, laser diffraction measurements are high quality and the technique may be useful when rapid measurements of filamentous cell clumps and pellets are required.