Single-crystal beta-MnO(2) nanotubes with diameters in the range 200-500 nm and lengths up to several micrometers were successfully prepared by a simple hydrothermal method through oxidizing MnSO(4) with NaClO(3) in the presence of poly(vinyl pyrrolidone) (PVP). It was found that the formation process of beta-MnO(2) nanotubes included two primary evolution stages over time: (1) the MnOOH nanoparticles initially formed in the hydrothermal system and anisotropic growth to nanorods and nanorod aggregates, and (2) the MnOOH nanorods transformed into beta-MnO(2) tubular structure and grown into beta-MnO(2) nanotubes due to continuous growth through a dissolution-recrystallization process eventually. Based on a series of experimental analysis, the formation mechanism of these nanostructures was discussed briefly. The present study has enlarged the family of nanotubes available and offers a possible new, general route to one-dimensional single-crystalline nanotubes of other materials.
Summary In Streptomyces, GlnR is an activator protein that activates nitrogen‐assimilation genes under nitrogen‐limiting conditions. However, less is known regarding the regulation of these genes under nitrogen‐rich conditions. We determined that the developmental regulator MtrA represses nitrogen‐assimilation genes in nitrogen‐rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP‐qPCR analysis. Transcriptional analysis indicated that, on nutrient‐rich medium, MtrA profoundly repressed expression of nitrogen‐associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.
dThe ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-␣), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.
The developmental life cycle of Streptomyces species includes aerial hyphae formation and spore maturation, two distinct developmental processes that are controlled, respectively, by two families of developmental regulatory genes, bld and whi. In this study, we show that the response regulator MtrA (SCO3013) is critical for normal development of aerial hyphae in S. coelicolor and related species. ΔmtrA, a deletion mutant of the response regulator gene mtrA, exhibited the bald phenotype typical of bld mutants defective in aerial mycelium formation, with formation either much delayed or absent depending on the culture medium. Transcriptional analysis indicated that MtrA activates multiple genes involved in formation of aerial mycelium, including chp, rdl, and ram genes, as well as developmental regulatory genes of the bld and whi families. However, the major regulatory gene bldD showed enhanced expression in ΔmtrA, suggesting it is repressed by MtrA. electrophoretic mobility shift assays indicated that MtrA binds upstream of several genes with altered expression in ΔmtrA, including bldD and whiI, and sequences similar to the consensus binding sequence for MtrA of another actinomycete, Mycobacterium tuberculosis, were found in the bound sites. A loosely conserved recognition sequence containing two short, direct repeats was identified for MtrA of S. coelicolor and was validated using mutational analysis. MtrA homologs are widely distributed among Streptomyces species, and as with S. coelicolor, deletion of the mtrA homologs sve_2757 from S. venezuelae and sli_3357 from S. lividans resulted in conditional bald morphology. Our study suggests a critical and conserved role for MtrA in Streptomyces development.
As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728, sco4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in Streptomyces. IMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.
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