The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria.     

Genome Sequence of a New Streptomyces coelicolor Generalized Transducing Bacteriophage, ?CAM

Streptomyces coelicolor is a model system for the study of Streptomyces, a genus of bacteria responsible for the production of many clinically important antibiotics. Here we report the genome sequence of ϕCAM, a new S. coelicolor generalized transducing bacteriophage, isolated from a soil sample originating from Lincolnshire, United Kingdom. Many open reading frames within ϕCAM shared high levels of similarity to a prophage from Salinispora tropica and a putative prophage in Streptomyces sp. strain C.     

Determination of ionophore antibiotics nactins produced by fecal Streptomyces from sheep

To investigate the correlation between fecal actinobacteria and host animals, Streptomyces was isolated from fresh faeces of healthy sheep and secondary metabolites were analyzed. The most frequently isolated strain S161 with antibiotic activity against bacteria and fungi were analyzed. The S161 showed the highest 99 % similarity to Streptomyces canus DSB17 based on the 16S rRNA gene sequence analysis. Metabolite analysis based on MS and NMR spectra showed that S161 produces nactins, cyclotetralactones derived from nonactic acid and homononactic acid as building units of ionophoretic character. Due to ionophores are antimicrobial compounds that are commonly fed to ruminant animals to improve feed efficiency, stable beneficial interactions between Streptomyces bacteria and vertebrates have been demonstrated.     

Streptomyces amritsarensis sp. nov., exhibiting broad-spectrum antimicrobial activity

A new actinobacterium strain, designated 2A^T, was isolated from a soil sample collected from Guru Nanak Dev University, Punjab (India) and characterized using a polyphasic taxonomic approach. It showed antimicrobial activity against various Gram-positive and Gram-negative bacteria including drug resistant bacteria and fungi. The strain had chemotaxononomic and morphological properties typical of the genus Streptomyces. The 16S rRNA gene sequence of the strain showed 99.9, 99.5 and 99.5 % similarity with Streptomyces flavotricini DSM 40152^T, Streptomyces toxytricini DSM 40178^T and Streptomyces globosus DSM 40815^T, respectively. This strain formed a coherent cluster with them and shared DNA–DNA homology of 37.6 ± 0.6, 34.4 ± 0.5 and 33.1 ± 0.4 % with type strains, S. flavotricini DSM 40152^T, S. globosus DSM 40815^T and S. toxytricini DSM 40178^T, respectively. Further, the strain was readily distinguished from the phylogenetic close relatives in a variety of morphological, physiological and biochemical properties. Based on the genotypic and phenotypic characteristics, it is proposed that strain 2A^T represents a novel species in the genus Streptomyces, for which the name Streptomyces amritsarensis sp. nov. is proposed, with the type strain 2A^T (=MTCC 11845^T=JCM 19660^T).     

A PKS/NRPS/FAS Hybrid Gene Cluster from Serratia plymuthica RVH1 Encoding the Biosynthesis of Three Broad Spectrum,Zeamine-Related Antibiotics

Serratia plymuthica strain RVH1, initially isolated from an industrial food processing environment, displays potent antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacterial pathogens. Isolation and subsequent structure determination of bioactive molecules led to the identification of two polyamino antibiotics with the same molecular structure as zeamine and zeamine II as well as a third, closely related analogue, designated zeamine I. The gene cluster encoding the biosynthesis of the zeamine antibiotics was cloned and sequenced and shown to encode FAS, PKS as well as NRPS related enzymes in addition to putative tailoring and export enzymes. Interestingly, several genes show strong homology to the pfa cluster of genes involved in the biosynthesis of long chain polyunsaturated fatty acids in marine bacteria. We postulate that a mixed FAS/PKS and a hybrid NRPS/PKS assembly line each synthesize parts of the backbone that are linked together post-assembly in the case of zeamine and zeamine I. This interaction reflects a unique interplay between secondary lipid and secondary metabolite biosynthesis. Most likely, the zeamine antibiotics are produced as prodrugs that undergo activation in which a nonribosomal peptide sequence is cleaved off.     

Identification of Streptomyces sp. nov. WH26 producing cytotoxic compounds isolated from marine solar saltern in China

A moderately halophilic actinomycetes strain, designated as WH26, was isolated from Weihai Solar Saltern in China. The identification of the strain WH26 was performed by its morphological characteristics, physiological and biochemical tests as well as phylogenetic analysis based on 16S rRNA sequence comparison. The results showed that the nucleotide sequence of the 16S rRNA gene (1,677 bp) of the strain WH26 exhibited close similarity (97–99 %) with other Streptomyces 16S rRNA genes and the strain WH26 was identified to belong to the genus Streptomyces. An ethyl acetate extraction of Streptomyces sp. nov. WH26 demonstrated significant cellular toxicity. Two compounds, 8-O-methyltetrangulol and naphthomycin A were isolated from the extract via silica gel column chromatography and HPLC. These two compounds showed potent cytotoxic activity against several human tumor cell lines including A549, HeLa, BEL-7402 and HT-29. The present studies suggest that moderately halophilic actinomycetes may be a novel biological source for the discovery of anticancer agents.     

Bioprospecting for genes involved in the production of chitosanases and microcystin-like compounds in Anabaena strains

Bioinformatic tools guided PCR amplification assays were employed for analyzing two Anabaena strains A. laxa and A. iyengarii which exhibited chitosanase activity, allelopathic and fungicidal activity. Sequencing of a 297 bp fragment obtained by amplification with primers directed towards mcy A gene (involved in the production of microcystins), revealed significant similarity with the condensation domain, while amplification with specific primers towards N-methyltransferase (NMT) domain showed 59% similarity with a homologous domain in a toxic strain of Microcystis aeruginosa. An amplified product of 172 bp obtained using specific primers derived from the coding region of chitinase (chi IS) gene in Streptomyces sp., showed 100% similarity with hydrogenbyrinic acid a, c-diamide cobaltochelatase gene in Anabaena, and significant similarity with chi IS gene of Streptomyces sp. under less stringent conditions. The 663 bp sequence obtained by employing specific primers for chitosanase (choA) derived from Mitsuaria chitosanitabida 3001 strain, showed 100% similarity with glycoside hydrolase family three domain like protein(s). This study is a first time report on the presence of homologues of chitosanase in cyanobacteria which can play a role in allelopathic activity exhibited by these oxygenic photosynthetic prokaryotes.     

Biochemical and Genetic Characterization of Propionicin T1, a New Bacteriocin from Propionibacterium thoenii

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii. This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii. The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA. The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.     

<Emphasis Type="Italic">Streptomyces gamaensis</Emphasis> sp. nov., a novel actinomycete with antifungal activity isolated from soil in Gama,Chad

During an investigation exploring potential sources of novel species and natural products, a novel actinomycete with antifungal activity, designated strain NEAU-Gz11^T, was isolated from a soil sample, which was collected from Gama, Chad. The isolate was found to have morphological and chemotaxonomic characteristics typical of members of the genus Streptomyces. 16S rRNA gene sequence similarity studies showed that strain NEAU-Gz11^T belongs to the genus Streptomyces with high sequence similarity to Streptomyces hiroshimensis JCM 4098^T (98.0 %). Similarities to other type strains of the genus Streptomyces were lower than 98.0 %. However, the physiological and biochemical characteristics and low levels of DNA–DNA relatedness could differentiate the isolate genotypically and phenotypically from S. hiroshimensis JCM 4098^T. Therefore, the strain is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces gamaensis sp. nov. is proposed. The type strain is NEAU-Gz11^T (=CGMCC 4.7304^T=DSM 101531^T).     

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.     

Biosynthesis of an engineered tautomycetin analogue via disruption of tmcK-encoding terminal decarboxylase in Streptomyces CK4412

Tautomycetin (TMC), produced by Streptomyces sp. CK4412, is an antifungal secondary metabolite with an unusual ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Recently, TMC was identified to possess additional biological functions including T cell-specific immunosuppressive and anti-cancer activities through differential inhibition of protein phosphatases, such as PP1, PP2A, and SHP2. These findings led us to isolate and characterize its entire biosynthetic and regulatory pathway gene cluster. In silico database comparisons revealed that the deduced products of two translationally coupled genes, a 666-bp tmcJ and a 1458-bp tmcK located on the 3′-terminus of the polyketide synthase gene, were found to have amino acid sequence homologies with putative bacterial decarboxylase genes. Targeted gene disruption of tmcK, but not tmcJ, from the Streptomyces sp. CK4412 chromosome resulted in production of a 5-deoxy-3″-carboxylic TMC. The tmcK mutant strain was functionally complemented using an integrative plasmid carrying tmcK and/or tmcJ–tmcK in order to restore TMC biosynthesis, a result suggesting that only TmcK is a functional TMC terminal decarboxylase. Unlike an authentic TMC, this engineered 5-deoxy-3″-carboxylic TMC analogue failed to show PP1 selectivity over PP2A, and it showed significantly reduced cytotoxicity against a human lung cancer cell line. These results imply that regio-specific modifications of TMC polyketide moiety, such as C3″-terminal carboxylation and/or C5-deketonization, could differentiate multiple biological activities in TMC produced from Streptomyces sp. CK4412.     

Precursor for biosynthesis of sugar moiety of doxorubicin depends on rhamnose biosynthetic pathway in Streptomyces peucetius ATCC 27952

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 contains a TDP-D-glucose 4,6-dehydratase gene, dnmM, that is putatively involved in the biosynthesis of daunosamine, but the gene contains a frameshift in the DNA sequence that would cause premature termination of translation. In pursuit of another TDP-D-glucose 4,6-dehydratase in S. peucetius, a homologue gene, rmbB, was found, whose deduced product exhibits high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was located within a putative rhamnose biosynthetic gene cluster at another locus in the genome. RmbB was verified to be a functional TDP-D-glucose 4,6-dehydratase by enzyme assay as it catalyzed the conversion of TDP-D-glucose into TDP-4-keto-6-deoxy-D-glucose. Inactivation of rmbB in the S. peucetius genome abolished the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restored the doxorubicin production. Hence, rmbB provides TDP-4-keto-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin.     

Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.     

Nature’s combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in <Emphasis Type="Italic">Streptomyces</Emphasis>

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.     

“Pseudo” γ-Butyrolactone Receptors Respond to Antibiotic Signals to Coordinate Antibiotic Biosynthesis

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal γ-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated “pseudo” GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.