Gut microbiota modulation by plant polyphenols in koi carp (Cyprinus carpio L.).

Plant polyphenol supplementation may improve fish health in aquaculture systems. To assess the potential benefits and function mechanism of plant polyphenols in aquaculture, fish were fed either basal feed (CON) or the basal feed supplemented with 500 mg/kg of curcumin (CUR), oligomeric proanthocyanidins (OPC), chlorogenic acid (CGA), or resveratrol (RES). After an 8-week feeding experiment, blood samples were used to analyze the concentrations of biochemical indices. Gut samples were collected to evaluate microbiota, short chain fatty acid (SCFA) levels, and gene expression. The results indicated that polyphenol administration reduced serum glucose and insulin. Lysozyme activity was enhanced by OPC and CGA, and superoxide dismutase activity was increased by CUR, OPC, and CGA. The gut microbial structure of the RES group was segregated from that of the CON, and the genus Bacteroides was identified as a potential biomarker in the CUR, CGA, and RES groups. Total gut SCFA increased in the CUR, CGA, and RES groups. A strong correlation was observed between Bacteroides and SCFA. In conclusion, dietary polyphenols have distinct anti-inflammatory, anti-oxidant, and anti-hyperglycemic activities that may be closely associated with their microbiota-modulation effects.

Transcriptional Regulator DasR Represses Daptomycin Production through Both Direct and Cascade Mechanisms in Streptomyces roseosporus.

Daptomycin, produced by Streptomyces roseosporus, is a clinically important cyclic lipopeptide antibiotic used for the treatment of human infections caused by drug-resistant Gram-positive pathogens. In contrast to most Streptomyces antibiotic biosynthetic gene clusters (BGCs), daptomycin BGC has no cluster-situated regulator (CSR) genes. DasR, a GntR-family transcriptional regulator (TR) widely present in the genus, was shown to regulate antibiotic production in model species S. coelicolor by binding to promoter regions of CSR genes. New findings reported here reveal that DasR pleiotropically regulates production of daptomycin and reddish pigment, and morphological development in S. roseosporus. dasR deletion enhanced daptomycin production and morphological development, but reduced pigment production. DasR inhibited daptomycin production by directly repressing dpt structural genes and global regulatory gene adpA (whose product AdpA protein activates daptomycin production and morphological development). DasR-protected regions on dptEp and adpAp contained a 16 nt sequence similar to the consensus DasR-binding site dre in S. coelicolor. AdpA was shown to target dpt structural genes and dptR2 (which encodes a DeoR-family TR required for daptomycin production). A 10 nt sequence similar to the consensus AdpA-binding site was found on target promoter regions dptAp and dptR2p. This is the first demonstration that DasR regulates antibiotic production both directly and through a cascade mechanism. The findings expand our limited knowledge of the regulatory network underlying daptomycin production, and will facilitate methods for construction of daptomycin overproducers.

Molecular characterization and biological functioning of interleukin-8 in Siberian sturgeon (Acipenser baeri).

Interleukin-8, otherwise known as CXCL8, is a CXC chemokine that plays a pivotal regulatory role in immune and inflammation responses of animals. Here, we identified an interleukin-8 homologue from Siberian sturgeon (Acipenser baeri), named AbIL-8, which belongs to the lineage 1 group of teleost fish IL-8s. The cDNA of Abil-8 is 1130 bp in length, containing a 5'- untranslated region (UTR) of 50 bp, a 3'- UTR of 783 bp, and an open reading frame (ORF) of 297 bp that encodes a protein consisting of 98 amino acids. The deduced AbIL-8 contained five cysteines, four of which are highly conserved, and an ELR motif typical of known mammalian CXC chemokines was also found preceding the CXC motif. Our phylogenetic analysis showed that AbIL-8 clustered with the CXCL8_L1 sequences from other teleosts, being clearly distinct from those of either birds or mammals. Abil-8 mRNA was constitutively expressed in all tested tissues and significantly up-regulated in the liver and spleen tissues by the bacteria Aernomas hydrophila. The in vitro experiment using primary spleen cells stimulated with heat-killed Aernomas hydrophila or lipopolysaccharide (LPS) revealed a similar expression pattern to that found in vivo, whereas stimulation on spleen cells with ß-glucan or polyI:C elicited negligible changes in levels of Abil-8 mRNA. Purified recombinant AbIL-8 not only exhibited chemotactic activity for lymphocytes and monocytes in peripheral blood leukocytes (PBLs) and, to a lesser extent, spleen cells, but also stimulated the proliferation of spleen cells at 10 ng/mLor above. Furthermore, intraperitoneal injection of rAbIL-8 also up-regulated the expression of immuno-related genes (IL-6, IgM and MHCIIß) at 24 h. Collectively, these results enhance our understanding of how IL-8 functions in the regulation of the immune responses in sturgeon.

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.

SAV742, a Novel AraC-Family Regulator from Streptomyces avermitilis, Controls Avermectin Biosynthesis, Cell Growth and Development.

Avermectins are useful anthelmintic antibiotics produced by Streptomyces avermitilis. We demonstrated that a novel AraC-family transcriptional regulator in this species, SAV742, is a global regulator that negatively controls avermectin biosynthesis and cell growth, but positively controls morphological differentiation. Deletion of its gene, sav_742, increased avermectin production and dry cell weight, but caused delayed formation of aerial hyphae and spores. SAV742 directly inhibited avermectin production by repressing transcription of ave structural genes, and also directly regulated its own gene (sav_742) and adjacent gene sig8 (sav_741). The precise SAV742-binding site on its own promoter region was determined by DNase I footprinting assay coupled with site-directed DNA mutagenesis, and 5-nt inverted repeats (GCCGA-n10/n12-TCGGC) were found to be essential for SAV742 binding. Similar 5-nt inverted repeats separated by 3, 10 or 15 nt were found in the promoter regions of target ave genes and sig8. The SAV742 regulon was predicted based on bioinformatic analysis. Twenty-six new SAV742 targets were identified and experimentally confirmed, including genes involved in primary metabolism, secondary metabolism and development. Our findings indicate that SAV742 plays crucial roles in not only avermectin biosynthesis but also coordination of complex physiological processes in S. avermitilis.

AvaR2, a pseudo γ-butyrolactone receptor homologue from Streptomyces avermitilis, is a pleiotropic repressor of avermectin and avenolide biosynthesis and cell growth.

Avermectins produced by Streptomyces avermitilis are effective anthelmintic agents. The autoregulatory signalling molecule that triggers avermectin biosynthesis is a novel butenolide-type molecule, avenolide, rather than common γ-butyrolactones (GBLs). We identified AvaR2, a pseudo GBL receptor homologue, as an important repressor of avermectin and avenolide biosynthesis and cell growth. AvaR2 directly repressed transcription of aveR (the ave cluster-situated activator gene), aco (a key gene for avenolide biosynthesis), its own gene (avaR2) and two other GBL receptor homologous genes (avaR1 and avaR3) by binding to their promoter regions. The aveR promoter had the highest affinity for AvaR2. A consensus 18 bp ARE (autoregulatory element)-like sequence was found in the AvaR2-binding regions of these five target genes. Eleven novel AvaR2 targets were identified, including genes involved in primary metabolism, ribosomal protein synthesis, and stress responses. AvaR2 bound and responded to endogenous avenolide and exogenous antibiotics jadomycin B (JadB) and aminoglycosides to modulate its DNA-binding activity. Our findings help to clarify the roles of pseudo GBL receptors as pleiotropic regulators and as receptors for new type autoregulator and exogenous antibiotic signal. A pseudo GBL receptor-mediated antibiotic signalling transduction system may be a common strategy that facilitates Streptomyces interspecies communication and survival in complex environments.

An extracytoplasmic function sigma factor, σ(25), differentially regulates avermectin and oligomycin biosynthesis in Streptomyces avermitilis.

σ(25) is an extracytoplasmic function (ECF) σ factor in the bacterium Streptomyces avermitilis that plays a differential regulatory role in avermectin and oligomycin biosynthesis. Gene deletion, complementation, and overexpression experiments showed that σ(25) inhibited avermectin production but promoted oligomycin production. σ(25) indirectly inhibited avermectin production by affecting the transcription of the pathway-specific activator gene aveR, whereas it directly activated oligomycin production by initiating transcription of the pathway-specific activator gene olmRI. The divergently transcribed genes smrAB are located upstream of sig25 and encode a putative two-component system (TCS). σ(25) was found to initiate its own transcription, and its expression was directly activated by SmrA. The precise SmrA-binding sites in the region upstream of sig25 were determined by DNase I footprinting assays and identified two direct repeat sequences CTGTGA-n5-CTGTGA, suggesting that SmrA regulates sig25 transcription by binding to these direct repeats. The deletion of smrAB had the similar effect on avermectin and oligomycin A production to the deletion of sig25, indicating that σ(25) and SmrAB function similarly in the regulation of antibiotic production. These findings helpfully clarify the regulation of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in S. avermitilis.