Microscopic and molecular genetic analyses of morphological development of the actinomycete Actinoplanes missouriensis.

The survival strategy of members of the bacterial genus Actinoplanes is fascinating from morphological and evolutionary perspectives. A brief motile phase is incorporated in the filamentous and resting stages of the life cycle of Actinoplanes missouriensis. Spores either lie dormant or swim under different external conditions. This review presents microscopic observations and molecular genetic analyses of A. missouriensis morphological development. Selected examples of the characterization of developmental genes and their products are also introduced.

CdgB Regulates Morphological Differentiation and Toyocamycin Production in Streptomyces diastatochromogenes 1628.

Bis (3',5')-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that controls several metabolic pathways in bacteria. In Streptomyces, c-di-GMP is associated with morphological differentiation, which is related to secondary metabolite production. In this study, we identified and characterized a diguanylate cyclase (DGC), CdgB, from Streptomyces diastatochromogenes 1628, which may be involved in c-di-GMP synthesis, through genetic and biochemical analyses. To further investigate the role of CdgB, the cdgB-deleted mutant strain Δ-cdgB and the cdgB-overexpressing mutant strain O-cdgB were constructed by genetic engineering. A phenotypic analysis revealed that the O-cdgB colonies exhibited reduced mycelium formation, whereas the Δ-cdgB colonies displayed wrinkled surfaces and shriveled mycelia. Notably, O-cdgB demonstrated a significant increase in the toyocamycin (TM) yield by 47.3%, from 253 to 374 mg/L, within 10 days. This increase was accompanied by a 6.7% elevation in the intracellular concentration of c-di-GMP and a higher transcriptional level of the toy cluster within four days. Conversely, Δ-cdgB showed a lower c-di-GMP concentration (reduced by 6.2%) in vivo and a reduced toyocamycin production (decreased by 28.9%, from 253 to 180 mg/L) after 10 days. In addition, S. diastatochromogenes 1628 exhibited a slightly higher inhibitory effect against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani compared to Δ-cdgB, but a lower inhibition rate than that of O-cdgB. The results imply that CdgB provides a foundational function for metabolism and the activation of secondary metabolism in S. diastatochromogenes 1628.

Morphologic Differentiation of the Exotic Parasitoid Eupelmus pulchriceps (Hymenoptera: Eupelmidae) in the Galapagos Archipelago.

The historical and geographical properties of the archipelagos allow a detailed study of species diversification, and phenotypic traits can indicate the extent of such processes. Eupelmus pulchriceps (Cameron, 1904) is an exotic species to the Galapagos archipelago, and generalist parasitoid that attacks a beetle species that consumes the seeds of the invasive shrub Leucaena leucocephala (Lam.) de Wit. Despite extensive sampling, the wasp is recorded only in Santa Cruz and San Cristobal islands of the Galapagos archipelago. Thus, using 112 female wasps, we compare body size, proportion, and allometric differentiations within and between the two islands. There were no body size differences between islands. A PerMANOVA indicates differences between the islands and a single differentiation between two localities of one island. Allometric differences between islands were not the same for all structures. These results are consistent with the greater distance between islands than between localities and suggest a differentiation process. The variables with allometric differentiation are associated with wings and ovipositor, possibly responding to different ecological pressures. It is interesting that this parasitoid, recently arrived at the archipelago, is already showing differentiation. Also, it is essential to monitor the behavior of these wasps in the archipelago, given their potential to access other species affecting the trophic interactions of the local biota.

The RasGEF MoCdc25 regulates vegetative growth, conidiation and appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae.

Ras guanine nucleotide exchange factors (RasGEFs) can trigger Ras GTPase activities and play important roles in controlling various cellular processes in eukaryotes. Recently, it has been exhibited that RasGEF Cdc25 regulates morphological differentiation and pathogenicity in several plant pathogenic fungi. However, the role of RasGEFs in Magnaporthe oryzae is largely unknown. In this study, we identified and functionally characterized a RasGEF gene MoCDC25 in M. oryzae, which is orthologous to Saccharomyces cerevisiae CDC25. Targeted gene deletion mutants (ΔMocdc25) were completely nonpathogenic and were severely impaired in hyphal growth, conidiation and appressorium formation. The mutants exhibited highly sensitive response to osmotic, cell wall integrity or oxidative stresses. MoCdc25 physically interacts with the MAPK scaffold Mst50 and the putative Cdc42GEF MoScd1 in yeast two-hybrid assays. Moreover, we found that MoCdc25 was involved in regulating the phosphorylation of the MAP kinases (Pmk1, Mps1, and Osm1). In addition, the intracellular cAMP content in hyphae of the ΔMocdc25 mutants was significantly reduced compared to the parent strain Ku80 and the defect of appressorium formation of the mutants could be partially restored by the supplement of exogenous cAMP. Taken together, we conclude that the RasGEF MoCdc25 regulates vegetative growth, conidiation, appressorium formation and pathogenicity via MAPK and cAMP response pathways in M. oryzae.

Role of fourteen XRE-DUF397 pairs from Streptomyces coelicolor as regulators of antibiotic production and differentiation. New players in a complex regulatory network.

Bacteria of the genus Streptomyces have a plethora of transcriptional regulators, among which the xenobiotic response element (XRE) plays an important role. In this organism, XRE regulators are often followed downstream by small proteins of unknown function containing a DUF397 domain. It has been proposed that XRE/DUF397 pairs constitute type II toxin-antitoxin (TA) systems. However, previous work carried out by our group has shown that one of these systems is a strong activator of antibiotic production in S. coelicolor and other Streptomyces species. In this work, we have studied the overexpression of fourteen XRE/DUF397 pairs present in the S. coelicolor genome and found that none behave as a type II TA system. Instead, they act as pleiotropic regulators affecting, in a dependent manner, antibiotic production and morphological differentiation on different culture media. After deleting, individually, six XRE/DUF397 pairs (those systems producing more notable phenotypic changes when overexpressed: SCO2246/45, SCO2253/52, SCO4176/77, SCO4678/79, SCO6236/35, and SCO7615/16), the pair SCO7615/16 was identified as producing the most dramatic differences as compared to the wild-type strain. The SCO7615/16 mutant had a different phenotype on each of the media tested (R2YE, LB, NMMP, YEPD, and MSA). In particular, on R2YE and YEPD media, a bald phenotype was observed even after 7 days, with little or no actinorhodin (ACT) production. Lower ACT production was also observed on LB medium, but the bacteria were able to produce aerial mycelium. On NMMP medium, the mutant produced a larger amount of ACT as compared with the wild-type strain.

Global Effects of the Developmental Regulator BldB in Streptomyces venezuelae.

In Streptomyces, the Bld (Bald) regulators control formation of the reproductive aerial hyphae. The functions of some of these regulators have been well characterized, but BldB has remained enigmatic. In addition to the bldB gene itself, Streptomyces venezuelae has 10 paralogs of bldB that sit next to paralogs of whiJ and abaA. Transcriptome sequencing (RNA-seq) revealed that loss of BldB function causes the dramatic transcriptional upregulation of the abaA paralogs and a novel inhibitor of sporulation, iosA, and that cooverexpression of just two of these genes, iosA and abaA6, was sufficient to recapitulate the bldB mutant phenotype. Further RNA-seq analysis showed that the transcription factor WhiJ9 is required for the activation of iosA seen in the bldB mutant, and biochemical studies showed that WhiJ9 mediates the activation of iosA expression by binding to direct repeats in the iosA-whiJ9 intergenic region. BldB and BldB9 hetero-oligomerize, providing a potential link between BldB and the iosA-whiJ9-bldB9 locus. This work greatly expands our overall understanding of the global effects of the BldB developmental regulator. IMPORTANCE To reproduce and disperse, the filamentous bacterium Streptomyces develops specialized reproductive structures called aerial hyphae. The formation of these structures is controlled by the bld (bald) genes, many of which encode transcription factors whose functions have been characterized. An exception is BldB, a protein whose biochemical function is unknown. In this study, we gain insight into the global effects of BldB function by examining the genome-wide transcriptional effects of deleting bldB. We identify a small set of genes that are dramatically upregulated in the absence of BldB. We show that their overexpression causes the bldB phenotype and characterize a transcription factor that mediates the upregulation of one of these target genes. Our results provide new insight into how BldB influences Streptomyces development.

Extracellular HSPA5 is autocrinally involved in the regulation of neuronal process elongation.

The molecular mechanisms by which neuronal processes grow are extremely complicated, involving fine-tuned regulation of extracellular and intracellular signals. It remains to be elucidated which molecules are contained in the regulation. Herein, we report for the first time that heat shock protein family A member 5 (HSPA5, also called immunoglobulin heavy chain binding endoplasmic reticulum [ER] protein [BiP]) is secreted from mouse primary dorsal neuronal ganglion (DRG) cells or neuronal cell line N1E-115, a frequently used neuronal differentiation model. Supporting these results, HSPA5 protein was co-localized not only with ER antigen KDEL but also with intracellular vesicles such as Rab11-positive secretory vesicles. Unexpectedly, addition of HSPA5 inhibited elongation of neuronal processes, whereas neutralization of extracellular HSPA5 with the antibodies elongated processes, characterizing extracellular HSPA5 as a negative regulator of neuronal differentiation. Treatment of cells with neutralizing antibodies for low-density lipoprotein receptor (LDLR) did not have significant effects on process elongation, whereas LDLR-related protein 1 (LRP1) antibodies promoted differentiation, implying that LRP1 may act as a receptor candidate for HSPA5. Interestingly, extracellular HSPA5 was greatly decreased following treatment with tunicamycin, an ER stress inducer, illustrating that the ability to form neuronal processes could be preserved, even under stress. These results suggest that neuronal HSPA5 itself is secreted to contribute to inhibitory effects on neuronal cell morphological differentiation and can be included on the list of extracellular signaling molecules negatively controlling differentiation.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and ß-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and ß-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

An overview on the two-component systems of Streptomyces coelicolor.

The two-component system (TCS) found in various organisms is a regulatory system, which is involved in the response by the organism to stimuli, thereby regulating the internal behavior of the cell. It is commonly found in prokaryotes and is an important signaling system in bacteria. TCSs are involved in the regulation of physiological and morphological differentiation of the industrially important microbes from the genus Streptomyces, which produce a vast array of bioactive secondary metabolites (SMs). Genetic engineering of TCSs can substantially increase the yield of target SMs, which is valuable for industrial-scale production. Research on TCS has mainly been completed in the model strain Streptomyces coelicolor. In this review, we summarize the recent advances in the functional identification and elucidation of the regulatory mechanisms of various TCSs in S. coelicolor, with a focus on their roles in the biosynthesis of important SMs.

Reproducible switching between a walled and cell wall-deficient lifestyle of actinomycetes using gradient agar plates.

The cell wall is a shape-defining structure that envelopes almost all bacteria, protecting them from biotic and abiotic stresses. Paradoxically, some filamentous actinomycetes have a natural ability to shed their cell wall under influence of hyperosmotic stress. These wall-deficient cells can revert to their walled state when transferred to a medium without osmoprotection but often lyse due to their fragile nature. Here, we designed plates with an osmolyte gradient to reduce cell lysis and thereby facilitating the transition between a walled and wall-deficient state. These gradient plates allow determining of the osmolyte concentration where switching takes place, thereby enabling careful and reproducible comparison between mutants affected by switching. Exploring these transitions could give valuable insights into the ecology of actinomycetes and their biotechnological applications.

Morphological and environmental differentiation as prezygotic reproductive barriers between parapatric and allopatric Campanula rotundifolia agg. cytotypes.

BACKGROUND AND AIMS: Reproductive isolation and local establishment are necessary for plant speciation. Polyploidy, the possession of more than two complete chromosome sets, creates a strong postzygotic reproductive barrier between diploid and tetraploid cytotypes. However, this barrier weakens between polyploids (e.g. tetraploids and hexaploids). Reproductive isolation may be enhanced by cytotype morphological and environmental differentiation. Moreover, morphological adaptations to local conditions contribute to plant establishment. However, the relative contributions of ploidy level and the environment to morphology have generally been neglected. Thus, the extent of morphological variation driven by ploidy level and the environment was modelled for diploid, tetraploid and hexaploid cytotypes of Campanula rotundifolia agg. Cytotype distribution was updated, and morphological and environmental differentiation was tested in the presence and absence of natural contact zones. METHODS: Cytotype distribution was assessed from 231 localities in Central Europe, including 48 localities with known chromosome counts, using flow cytometry. Differentiation in environmental niche and morphology was tested for cytotype pairs using discriminant analyses. A structural equation model was used to explore the synergies between cytotype, environment and morphology. KEY RESULTS: Tremendous discrepancies were revealed between the reported and detected cytotype distribution. Neither mixed-ploidy populations nor interploidy hybrids were detected in the contact zones. Diploids had the broadest environmental niche, while hexaploids had the smallest and specialized niche. Hexaploids and spatially isolated cytotype pairs differed morphologically, including allopatric tetraploids. While leaf and shoot morphology were influenced by environmental conditions and polyploidy, flower morphology depended exclusively on the cytotype. CONCLUSIONS: Reproductive isolation mechanisms vary between cytotypes. While diploids and polyploids are isolated postzygotically, the environmental niche shift is essential between higher polyploids. The impact of polyploidy and the environment on plant morphology implies the adaptive potential of polyploids, while the exclusive relationship between flower morphology and cytotype highlights the role of polyploidy in reproductive isolation.

Nitric Oxide Signaling for Aerial Mycelium Formation in Streptomyces coelicolor A3(2) M145.

Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes. IMPORTANCE Eukaryotic and prokaryotic cells utilize nitric oxide (NO) to regulate physiological functions. Besides its role as a producer of different bioactive substances, Streptomyces is suggested to be involved in mycelial development regulated by endogenously produced NO. However, the regulatory mechanisms are unclear. In this study, we proposed that NO signaling is involved in aerial mycelium formation in S. coelicolor A3(2) M145. NO serves as a signaling molecule for the regulation of intracellular cyclic 3',5'-diguanylate (c-di-GMP) levels, resulting in aerial mycelium formation controlled by a c-di-GMP receptor, BldD. As the abundant production of valuable secondary metabolites is closely related to the initiation of morphological differentiation in Streptomyces, NO may provide value for application in industrial fermentation by serving as a tool for regulating secondary metabolism.

Global Regulator AdpA_1075 Regulates Morphological Differentiation and Ansamitocin Production in Actinosynnema pretiosum subsp. auranticum.

Actinosynnema pretiosum is a well-known producer of maytansinoid antibiotic ansamitocin P-3 (AP-3). Growth of A. pretiosum in submerged culture was characterized by the formation of complex mycelial particles strongly affecting AP-3 production. However, the genetic determinants involved in mycelial morphology are poorly understood in this genus. Herein a continuum of morphological types of a morphologically stable variant was observed during submerged cultures. Expression analysis revealed that the ssgA_6663 and ftsZ_5883 genes are involved in mycelial aggregation and entanglement. Combing morphology observation and morphology engineering, ssgA_6663 was identified to be responsible for the mycelial intertwining during liquid culture. However, down-regulation of ssgA_6663 transcription was caused by inactivation of adpA_1075, gene coding for an AdpA-like protein. Additionally, the overexpression of adpA_1075 led to an 85% increase in AP-3 production. Electrophoretic mobility shift assays (EMSA) revealed that AdpA_1075 may bind the promoter regions of asm28 gene in asm gene cluster as well as the promoter regions of ssgA_6663. These results confirm that adpA_1075 plays a positive role in AP-3 biosynthesis and morphological differentiation.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

MacRS controls morphological differentiation and natamycin biosynthesis in Streptomyces gilvosporeus F607.

Streptomyces gilvosporeus F607 produces large amounts of natamycin in a process regulated by multiple networks, including two-component systems (TCSs). The macR and macS genes, which are annotated as rs12540 and rs12545, respectively, in S. gilvosporeus F607, affect natamycin biosynthesis and sporulation. The findings of this study indicate that deletion of macRS from S. gilvosporeus F607 prevents the production of natamycin, delays spore formation (according to scanning electron microscopy), and results in aerial hyphae lacking compartments separated by septa (according to transmission electron microscopy). Real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that the expression levels of natamycin biosynthesis-related genes and genes essential for septum formation during sporulation were affected in the ΔmacRS mutant strain. Molecular simulations and electrophoretic mobility shift assays (EMSAs) suggested MacR not only interacted with the intergenic region of sgnM and sgnR, but also with the promoter of penicillin-binding protein gene ftsL required for cell division. sgnR promoter was presumed to be the binding target of MacR based on the RT-qPCR results. MacR had different affinity with two binding sites: one was located at ftsL promoter region with a perfect inverted repeats 'TGAGTACGCGTACTCA', the other was located at the presumed sgnR promoter with an imperfect inverted repeats 'TGAAGGTGCTGGACTCA'. We propose a hypothesis of a three-level regulatory pathway based on pleiotropic transcriptional regulator MacR and its target genes sgnR and ftsL; the pathway activates natamycin biosynthesis and influences septum development via direct and indirect effects in S. gilvosporeus F607.

AdpA, a developmental regulator, promotes ε-poly-L-lysine biosynthesis in Streptomyces albulus.

BACKGROUND: AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces. RESULTS: In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus. CONCLUSIONS: Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

Protein X0P338, a GntR-type pleiotropic regulator for morphological differentiation and secondary metabolites production in Streptomyces diastatochromogenes 1628.

The nucleoside antibiotic, toyocamycin (TM) exhibits excellent potent activity against several phytopathogenic fungi. Despite its importance, little is known about key factors regulating TM biosynthesis and morphological differentiation in Streptomyces diastatochromogenes 1628. Based on proteomics data obtained from the analysis between wild-type (WT) S. diastatochromogenes 1628 strain and mutant strain 1628-T62 having a low yield of TM, we observed that the differentially expressed protein, X0P338, which was proposed to be a regulator of the GntR-family, exhibited a higher expression level in S. diastatochromogenes 1628. Therefore, in this study, to explore whether protein X0P338 was involved in morphological differentiation and biosynthesis of secondary metabolites, especially TM, the gene called the gntRsd -encoding protein X0P338 was cloned and overexpressed in WT strain 1628 and mutant strain 1628-T62, respectively. The results indicated that the overexpression of gntRsd enhanced TM production in both strain 1628 (120.6 mg/L vs. 306.6 mg/L) and strain 1628-T62 (15.6 mg/L vs. 258.9 mg/L). Besides, the overexpression of gntRsd had positive and negative effects on morphological differentiation in strain 1628 and strain 1628-T62, respectively. The results also showed opposite effects on tetraene macrolide production during the overexpression of gntRsd in strain 1628 and strain 1628-T62. Moreover, transcription levels of genes involved in morphological differentiation and secondary metabolites production were affected by the overexpression of gntRsd gene, both in strain 1628 and strain 1628-T62. These results confirm that X0P338 as a GntR-type pleiotropic regulator that regulates the morphological differentiation and biosynthesis of secondary metabolites, and especially has a positive effect on TM biosynthesis.

Zinc-Responsive Regulator Zur Regulates Zinc Homeostasis, Secondary Metabolism, and Morphological Differentiation in Streptomyces avermitilis.

Zinc is an essential cofactor for many metal enzymes and transcription regulators. Zn2+ availability has long been known to affect antibiotic production and morphological differentiation of Streptomyces species. However, the molecular mechanism whereby zinc regulates these processes remains unclear. We investigated the regulatory roles of the zinc-sensing regulator Zur in Streptomyces avermitilis. Our findings demonstrate that Zur plays an essential role in maintaining zinc homeostasis by repressing the expression of the zinc uptake system ZnuACB and alternative non-zinc-binding ribosomal proteins and promoting the expression of zinc exporter ZitB. Deletion of the zur gene resulted in decreased production of avermectin and oligomycin and delayed morphological differentiation, and these parameters were restored close to wild-type levels in a zur-complemented strain. Zur bound specifically to Zur box in the promoter regions of avermectin pathway-specific activator gene aveR, oligomycin polyketide synthase gene olmA1, and filipin biosynthetic pathway-specific regulatory genes pteR and pteF. Analyses by reverse transcription quantitative PCR and luciferase reporter systems indicated that Zur directly activates the transcription of these genes, i.e., that Zur directly activates biosynthesis of avermectin and oligomycin. Zur positively regulated morphological development by repressing the transcription of differentiation-related genes ssgB and minD2. Our findings, taken together, demonstrate that Zur in S. avermitilis directly controls zinc homeostasis, biosynthesis of avermectin and oligomycin, and morphological differentiation. IMPORTANCE Biosynthesis of secondary metabolites and morphological differentiation in bacteria are affected by environmental signals. The molecular mechanisms whereby zinc availability affects secondary metabolism and morphological differentiation remain poorly understood. We identified several new target genes of the zinc response regulator Zur in Streptomyces avermitilis, the industrial producer of avermectin. Zur was found to directly and positively control avermectin production, oligomycin production, and morphological differentiation in response to extracellular Zn2+ levels. Our findings clarify the regulatory functions of Zur in Streptomyces, which involve linking environmental Zn2+ status with control of antibiotic biosynthetic pathways and morphological differentiation.

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Gobiids are widespread invasive species, with many species from this group usually invade into the same ecosystem simultaneously. Understanding the mechanisms underlying the coexi-stence of different gobiid species in the sympatric habitats is a key issue in fish invasion ecology. Incorporating morphological analyses, spatial distribution survey, and trophic analyses, we examined the coexistence strategy of Mugilogobius myxodermus and related species (the earlier invaders) in Dianchi Lake, Yunnan, China. Our results showed significant differences in morphology and spatial distribution among the four invasive gobies species (i.e., M. myxodermus, Micropercops swinhonis, Rhinogobius giurinus and Rhinogobius cliffordpopei). The spatial niche index of M. myxodermus was the highest. Food composition between M. myxodermus and other gobies was significantly different, with the former mainly feeding on Chydorus ovalis and Cypris sp. The trophic diversity index of M. myxodermus was the highest. Overall, we found that morphological differences, spatial niche diffe-rentiation, and trophic niche differentiation contributed to the coexistence of the gobies in Dianchi Lake, which could help M. myxodermus reduce interspecific competition. Importantly, the feeding strategy is the key factor determining population size and habitas of M. myxodermus during their competition with the other gobies, and finally contributing to the dominant position in the study area.

Parallel evolution of morphological traits and body shape in littoral and pelagic brook charr, Salvelinus fontinalis, along a gradient of interspecific competition.

The parallel evolution of similar ecotypes in response to comparable environmental conditions is believed to reveal the importance of divergent selection in phenotypic diversifying processes. Systems characterized by the presence of multiple replicate populations expressing resource polymorphism thus provide an ideal opportunity to address the occurrence and factors affecting the parallel evolution of ecotypes. Previous studies have shown that brook charr (Salvelinus fontinalis) exhibit resource polymorphism in some Canadian Shield lakes, where a littoral ecotype feeds mainly on zoobenthos and a pelagic ecotype feeds mostly on zooplankton. Using morphological traits and geometric morphometric analyses on 18 native brook charr populations, we explicitly tested (i) whether brook charr ecotypes show parallel evolution across populations (i.e. the same morphological traits discriminate ecotypes among lakes) and (ii) whether interspecific competition decreases the amplitude of morphological differentiation between ecotypes, if any, because brook charr experience some level of competitive exclusion from the littoral habitat in the presence of creek chub or white sucker. We observed a low level of parallel evolution, where the littoral ecotype was overall stouter with longer fins and smaller eyes than the pelagic ecotype. Interspecific competition had no clear impacts on the amplitude of morphological differentiation. We also observed that inter-lake morphological differences are greater than between ecotypes within lakes, suggesting an important effect of local environmental factors on population morphology. Early-stage of diversification as well as phenotypic plasticity and morphological integration could explain why resource polymorphism is still subtle in brook charr populations.