Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

<p>Abstract</p> <p>Background</p> <p>The chromosome of <it>Streptomyces </it>has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of <it>Streptomyces avermitilis</it>, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in <it>Streptomyces</it>.</p> <p>Results</p> <p>Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable.</p> <p>Conclusions</p> <p>Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in <it>S. avermitilis </it>by non-homologous recombination. The finding of an inner deletion involving in the central region of <it>S. avermitilis </it>chromosome suggests that the entire <it>Streptomyces </it>chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends.</p

Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis

The bacterium Streptomyces avermitilis is an industrial-scale producer of avermectins, which are important anthelmintic agents widely used in agriculture, veterinary medicine, and human medicine. During the avermectin fermentation process, S. avermitilis is exposed to organic peroxides generated by aerobic respiration. We investigated the role of MarR-family transcriptional regulator OhrR in oxidative stress response and avermectin production in S. avermitilis. The S. avermitilis genome encodes two organic hydroperoxide resistance proteins: OhrB1 and OhrB2. OhrB2 is the major resistance protein in organic peroxide stress responses. In the absence of organic peroxide, the reduced form of OhrR represses the expression of ohrB2 gene by binding to the OhrR box in the promoter region. In the presence of organic peroxide, the oxidized form of OhrR dissociates from the OhrR box and the expression of ohrB2 is increased by derepression. OhrR also acts as a repressor to regulate its own expression. An ohrR-deletion mutant (termed DohrR) displayed enhanced avermectin production. Our findings demonstrate that OhrR in S. avermitilis represses avermectin production by regulating the expression of pathway-specific regulatory gene aveR. OhrR also plays a regulatory role in glycolysis and the pentose phosphate (PP) pathway by negatively controlling the expression of pykA2 and ctaB/tkt2-tal2-zwf2-opcA2-pgl

Графический дизайн как визуальный язык межкультурного взаимодействия

This article describes how visual graphics language as a sign system can be in contact with the audience, overcoming the language barrier. In terms of graphic design it can be available to transfer information, and even affect the viewer, causing artistic and emotional reflection.Эта статья о том, как визуальный язык графики в виде знаковой символики может входить в контакт со зрителем, преодолевая языковый барьер. На языке графического дизайна можно доступно передать информацию и даже воздействовать на зрителя, вызывая при этом художественно-эмоциональные образы

Fine-grained sublacustrine fan deposits and their significance in shale oil reservoirs in the Lucaogou Formation in the Jimsar Sag, Junggar Basin

Objective The Jimsar Sag of the Junggar Basin is representative of successful shale oil exploration in China. However, the sedimentary facies in the Permian Lucaogou Formation hosting shale oil has long been disputed. Previous studies proposed two contradictory environments of shallow lakes and semideep to deep lakes in the interior lacustrine basin and identified sandstone interlays as deltaic distal sandbar deposition and beach and bar deposition. Methods The study combined core observations with analyses of sandstone granularity, logging facies, and reservoir properties to identify the development of sublacustrine fans in a semi-deep to deep lake background. Results Fine-grained turbidity currents played a significant role in the transportation and deposition of sediment in these fans. The lithofacies observed included lenticular-wave bedded muddy siltstone to siltstone (dominant type), parallel bedded siltstone, graded siltstone, and massive-graded bedded siltstone to fine-grained sandstone. The cumulative probability granularity curves exhibited smooth upper arches with a high suspension population, and the cross-spots on the C-M diagram were located in the grade suspension zone. The sublacustrine fans consisted of sedimentary microfacies of sheet-like lobes and gravity flow channel levees, showing fining- and coarsening-upwards successions, respectively. These fans contained numerous interlayers of siltstone and fine-grained sandstone, as well as terrigenous detrital particles that influenced the mineral content and enhanced shale oil reservoir properties by promoting the growth of solution pores. Conclusion The study concluded that sublacustrine fan deposits are thin-bedded and fine-grained, and they occur frequently. It also determined that desert reservoirs formed in the areas where sublacustrine fans were deposited, which has significant implications for shale oil exploration in the study area

An Alternative σ Factor, σ8, Controls Avermectin Production and Multiple Stress Responses in Streptomyces avermitilis

Alternative σ factors in bacteria redirect RNA polymerase to recognize alternative promoters, thereby facilitating coordinated gene expression necessary for adaptive responses. The gene sig8 (sav_741) in Streptomyces avermitilis encodes an alternative σ factor, σ8, highly homologous to σB in Streptomyces coelicolor. Studies reported here demonstrate that σ8 is an important regulator of both avermectin production and stress responses in S. avermitilis. σ8 inhibited avermectin production by indirectly repressing expression of cluster-situated activator gene aveR, and by directly initiating transcription of its downstream gene sav_742, which encodes a direct repressor of ave structural genes. σ8 had no effect on cell growth or morphological differentiation under normal growth conditions. Growth of a sig8-deletion mutant was less than that of wild-type strain on YMS plates following treatment with heat, H2O2, diamide, NaCl, or KCl. sig8 transcription was strongly induced by these environmental stresses, indicating response by σ8 itself. A series of σ8-dependent genes responsive to heat, oxidative and osmotic stress were identified by EMSAs, qRT-PCR and in vitro transcription experiments. These findings indicate that σ8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes. Six σ8-binding promoter sequences were determined and consensus binding sequence BGVNVH-N15-GSNNHH (B: C, T or G, V: A, C or G, S: C or G, H: A, C or T, N: any nucleotide) was identified, leading to prediction of the σ8 regulon. The list consists of 940 putative σ8 target genes, assignable to 17 functional groups, suggesting the wide range of cellular functions controlled by σ8 in S. avermitilis

AvaR1, a Butenolide-Type Autoregulator Receptor in Streptomyces avermitilis, Directly Represses Avenolide and Avermectin Biosynthesis and Multiple Physiological Responses

Avermectins are commercially important anthelmintic antibiotics produced by Streptomyces avermitilis. The homologous TetR-family transcriptional regulators AvaR1 and AvaR2 in this species were identified previously as receptors of avenolide, a novel butenolide-type autoregulator signal required for triggering avermectin biosynthesis. AvaR2 was found to be an important pleiotropic regulator in repression of avermectin and avenolide production and cell growth, whereas the regulatory role of AvaR1 remains unclear. Investigation of AvaR1 function in the present study showed that it had no effect on cell growth or morphological differentiation, but inhibited avenolide and avermectin production mainly through direct repression of aco (the key enzyme gene for avenolide biosynthesis) and aveR (the cluster-situated activator gene). AvaR1 also directly repressed its own gene (avaR1) and two adjacent homologous genes (avaR2 and avaR3). Binding sites of AvaR1 on these five target promoter regions completely overlapped those of AvaR2, leading to the same consensus binding motif. However, AvaR1 and AvaR2 had both common and exclusive target genes, indicating that they cross-regulate diverse physiological processes. Ten novel identified AvaR1 targets are involved in primary metabolism, stress responses, ribosomal protein synthesis, and cyclic nucleotide degration, reflecting a pleiotropic role of AvaR1. Competitive EMSAs and GST pull-down assays showed that AvaR1 and AvaR2 competed for the same binding regions, and could form a heterodimer and homodimers, suggesting that AvaR1 and AvaR2 compete and cooperate to regulate their common target genes. These findings provide a more comprehensive picture of the cellular responses mediated by AvaR1 and AvaR2 regulatory networks in S. avermitilis

Relationships between SAV576 and SAV577.

<p>(<b>A</b>) Comparison of DNA-binding affinity of SAV576 and SAV577 with probes 1 and 577p. Each lane contained 0.15 nM labeled probe. White arrows: free probes. Black arrows: DNA-protein complexes. (<b>B</b>) Competitive EMSAs of probe 1 with His₆-SAV576 and His₆-SAV577 proteins. End-labeled probe 1 was incubated with the indicated concentrations of His₆-SAV576 or His₆-SAV577. Lanes 1 and 1′: control reactions (no protein added). Diagonal arrows: two complexes in lanes 3 and 3′. (<b>C</b>) GST pull-down assays of SAV576 and SAV577 from <i>E. coli</i> whole cell lysate. GST- and His₆-tagged proteins were co-expressed in <i>E. coli</i>, lysed by sonication, and subjected to GST pull-down assay and Western blotting using anti-GST and anti-His antibodies, respectively. Lanes 1, cell lysate before IPTG induction. Lanes 2, cell lysate after induction. Lanes 3, GST pull-down.</p

Two Adjacent and Similar TetR Family Transcriptional Regulator Genes, <i>SAV577</i> and <i>SAV576</i>, Co-Regulate Avermectin Production in <i>Streptomyces avermitilis</i>

<div><p><i>Streptomyces avermitilis</i> is an important bacterial species used for industrial production of avermectins, a family of broad-spectrum anthelmintic agents. We previously identified the protein SAV576, a TetR family transcriptional regulator (TFR), as a downregulator of avermectin biosynthesis that acts by controlling transcription of its major target gene <i>SAV575</i> (which encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and <i>ave</i> genes. <i>SAV577</i>, another TFR gene, encodes a SAV577 protein that displays high amino acid homology with SAV576. In this study, we examined the effect of SAV577 on avermectin production and the relationships between SAV576 and SAV577. SAV577 downregulated avermectin biosynthesis indirectly, similarly to SAV576. SAV576 and SAV577 both directly repressed <i>SAV575</i> transcription, and reciprocally repressed each other's expression. <i>SAV575</i> transcription levels in various <i>S. avermitilis</i> strains were correlated with avermectin production levels. DNase I footprinting and electrophoretic mobility shift assays indicated that SAV576 and SAV577 compete for the same binding regions, and that DNA-binding affinity of SAV576 is much stronger than that of SAV577. GST pull-down assays revealed no direct interaction between the two proteins. Taken together, these findings suggest that SAV577 regulates avermectin production in <i>S. avermitilis</i> by a mechanism similar to that of SAV576, and that the role of SAV576 is dominant over that of SAV577. This is the first report of two adjacent and similar TFR genes that co-regulate antibiotic production in <i>Streptomyces.</i></p></div