Landscape of Post-Transcriptional tRNA Modifications in Streptomyces albidoflavus J1074 as Portrayed by Mass Spectrometry and Genomic Data Mining.

Actinobacterial genus Streptomyces (streptomycetes) represents one of the largest cultivable group of bacteria famous for their ability to produce valuable specialized (secondary) metabolites. Regulation of secondary metabolic pathways inextricably couples the latter to essential cellular processes that determine levels of amino acids, carbohydrates, phosphate, etc. Post-transcriptional tRNA modifications remain one of the least studied aspects of streptomycete physiology, albeit a few of them were recently shown to impact antibiotic production. In this study, we describe the diversity of post-transcriptional tRNA modifications in model strain Streptomyces albus (albidoflavus) J1074 by combining mass spectrometry and genomic data. Our results show that J1074 can produce more chemically distinct tRNA modifications than previously thought. An in silico approach identified orthologs for enzymes governing most of the identified tRNA modifications. Yet, genetic control of certain modifications remained elusive, suggesting early divergence of tRNA modification pathways in Streptomyces from the better studied model bacteria, such as Escherichia coli and Bacillus subtilis. As a first point in case, our data point to the presence of a non-canonical MiaE enzyme performing hydroxylation of prenylated adenosines. A further finding concerns the methylthiotransferase MiaB, which requires previous modification of adenosines by MiaA to i6A for thiomethylation to ms2i6A. We show here that the J1074 ortholog, when overexpressed, yields ms2A in a ΔmiaA background. Our results set the working ground for and justify a more detailed studies of biological significance of tRNA modification pathways in streptomycetes. IMPORTANCE Post-transcriptional tRNA modifications (PTTMs) play an important role in maturation and functionality of tRNAs. Little is known about tRNA modifications in the antibiotic-producing actinobacterial genus Streptomyces, even though peculiar tRNA-based regulatory mechanisms operate in this taxon. We provide a first detailed description of the chemical diversity of PTTMs in the model species, S. albidoflavus J1074, and identify most plausible genes for these PTTMs. Some of the PTTMs are described for the first time for Streptomyces. Production of certain PTTMs in J1074 appears to depend on enzymes that show no sequence similarity to known PTTM enzymes from model species. Our findings are of relevance for interrogation of genetic basis of PTTMs in pathogenic actinobacteria, such as M. tuberculosis.

Insights into the Biological Properties of Ligands and Identity of Operator Site for LanK Protein Involved in Landomycin Production.

LanK is a TetR type regulatory protein that coordinates the late steps of the biosynthesis of the landomycin family of antitumor angucyclic polyketides and their export from the cells of Streptomyces cyanogenus S136. We recently described the structure of LanK and showed that it is the carbohydrate portion of the landomycins that is responsible for abrogating the repressing effect of LanK on landomycin production and export. The effect has been established in a series of in vitro tests using synthetic analogs of the landomycin carbohydrate chains. Whether such synthetic compounds would function as effector molecules for LanK under in vivo conditions remained unknown. Furthermore, the location and identity of LanK operator sites within the lanK-lanJ intergenic region (lanKJp) was unknown. Here we report that methoxyphenyl analogs of tri- and hexasaccharide chains of landomycins cannot function as LanK ligands when applied externally to the reporter strain. The lability of these compounds to cellular media and/or their poor penetration into the cells could explain our observations. The LanK operator site has been mapped to a 14-bp region of lanKJp that includes a plausible -35 site upstream of the lanK start codon in a series of electrophoretic DNA mobility shift assays. This opens the door to studies of the DNA-LanK interaction at a single nucleotide resolution level.

Genetic approaches to improve clorobiocin production in Streptomyces roseochromogenes NRRL 3504.

Streptomyces roseochromogenes NRRL 3504 is best known as a producer of clorobiocin, a DNA replication inhibitor from the aminocoumarin family of antibiotics. This natural product currently draws attention as a promising adjuvant for co-application with other antibiotics against Gram-negative multidrug-resistant pathogens. Herein, we expand the genetic toolkit for NRRL 3504 by showing that a set of integrative and replicative vectors, not tested previously for this strain, could be conjugally transferred at high frequency from Escherichia coli to NRRL 3504. Using this approach, we leverage a cumate-inducible expression of cluster-situated regulatory gene novG to increase clorobiocin titers by 30-fold (up to approximately 200 mg/L). To our best knowledge, this is the highest level of clorobiocin production reported so far. Our findings set a working ground for further improvement of clorobiocin production as well as for the application of genetic methods to illuminate the cryptic secondary metabolome of NRRL 3504. Key Points • Efficient system for conjugative transfer of plasmids into NRRL 3504 was developed. • Expression of regulatory genes in NRRL 3504 led to increase in clorobiocin titer. • Secondary metabolome of NRRL 3504 becomes an accessible target for genetic manipulations using the expanded vector set and improved intergeneric conjugation protocol.

Heterologous Expression Reveals Ancient Properties of Tei3­A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus­the producer of the last-resort-drug teicoplanin­has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.

Genetically engineered rpsL merodiploidy impacts secondary metabolism and antibiotic resistance in Streptomyces.

Certain point mutations within gene for ribosomal protein S12, rpsL, are known to dramatically change physiological traits of bacteria, most prominently antibiotic resistance and production of various metabolites. The rpsL mutants are usually searched among spontaneous mutants resistant to aminoglycoside antibiotics, such as streptomycin or paromomycin. The shortcomings of traditional selection are as follows: random rpsL mutants may carry undesired genome alterations; many rpsL mutations cannot be isolated because they are either not associated with increased antibiotic resistance or non-viable in the absence of intact rpsLWT gene. Introduction of mutant rpsL alleles in the rpsLWT background can be used to circumvent these obstacles. Here we take the latter approach and report the generation and properties of a set of stable rpsL merodiploids for Streptomyces albus J1074. We identified several rpsL alleles that enhance endogenous and heterologous antibiotic production by this strain and show that rpsLWTrpsLK88E merodiploid displays increased streptomycin resistance. We further tested several promising rpsL alleles in two more strains, Streptomyces cyanogenus S136 and Streptomyces ghanaensis ATCC14672. In S136, plasmid-borne rpsLK88E+P91S and rpsLK88R led to elevated landomycin production; no changes were detected for ATCC14672 merodiploids. Our data outline the prospects for and limitations to rpsL merodiploids as a tool for rapid enhancement of secondary metabolism in Streptomyces.

The Use of the Rare TTA Codon in Streptomyces Genes: Significance of the Codon Context?

Streptomycetes, Gram-positive bacteria with huge and GC-rich genomes provide an ample example of codon usage bias taken to the extreme. Particularly, in all sequenced to date streptomycete genomes leucyl codon TTA is the rarest one. It is present (usually once or twice) in 70-200 out of 7000-8000 coding sequences that make up a typical streptomycete genome. tRNALeu UAA of streptomycetes, encoded by the bldA gene, has been shown to be present in mature form only after the onset of morphological differentiation and activation of secondary metabolism. Consequently, during the early stages of cell growth, the translation of genes carrying the TTA codon can be interrupted due to the absence of tRNALeu UAA. Several reports show that mutations of TTA to synonymous codons in certain genes indeed relieve their expression from bldA dependence. However, the deletion of bldA does not always arrest the expression of TTA-containing genes. The nucleotides T/C downstream of TTA were suggested, in 2002, to favor TTA mistranslation. We tested this hypothesis using sizable datasets derived from individual Streptomyces genome and a subset of TTA+ genes for secondary metabolism known for their active expression. Our results revealed nucleotide biases downstream of NNA codons family, such as the preference for C and the avoidance of A. Yet, none of the observed biases was sufficient to claim a special case for TTA codon. Hence, the issue of codon context and TTA codon mistranslation in Streptomyces deserves further elaboration.

Gene miaA for post-transcriptional modification of tRNAXXA is important for morphological and metabolic differentiation in Streptomyces.

Members of actinobacterial genus Streptomyces possess a sophisticated life cycle and are the deepest source of bioactive secondary metabolites. Although morphogenesis and secondary metabolism are subject to transcriptional co-regulation, streptomycetes employ an additional mechanism to initiate the aforementioned processes. This mechanism is based on delayed translation of rare leucyl codon UUA by the only cognate tRNALeu UAA (encoded by bldA). The bldA-based genetic switch is an extensively documented example of translational regulation in Streptomyces. Yet, after five decades since the discovery of bldA, factors that shape its function and peculiar conditionality remained elusive. Here we address the hypothesis that post-transcriptional tRNA modifications play a role in tRNA-based mechanisms of translational control in Streptomyces. Particularly, we studied two Streptomyces albus J1074 genes, XNR_1074 (miaA) and XNR_1078 (miaB), encoding tRNA (adenosine(37)-N6)-dimethylallyltransferase and tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase respectively. These enzymes produce, in a sequential manner, a hypermodified ms2 i6 A37 residue in most of the A36-A37-containing tRNAs. We show that miaB and especially miaA null mutant of S. albus possess altered morphogenesis and secondary metabolism. We provide genetic evidence that miaA deficiency impacts translational level of gene expression, most likely through impaired decoding of codons UXX and UUA in particular.

Teicoplanin biosynthesis: unraveling the interplay of structural, regulatory, and resistance genes.

Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.

Gene ssfg_01967 (miaB) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.

Landomycin biosynthesis and its regulation in Streptomyces.

This mini-review is centered on genetic aspects of biosynthesis of landomycins (La), a family of angucycline polyketides. From the very discovery in the 1990s, La were noted for unusual structure and potent anticancer properties. La are produced by a few actinobacteria that belong to genus Streptomyces. Biochemical logic behind the production of La aglycon and glycoside halves and effects of La on mammalian cells have been thoroughly reviewed in 2009-2012. Yet, the genetic diversity of La biosynthetic gene clusters (BGCs) and regulation of their production were not properly reviewed since discovery of La. Here, we aim to fill this gap by focusing on three interrelated topics. First, organization of known La BGCs is compared. Second, up-to-date scheme of biosynthetic pathway to landomycin A (LaA), the biggest (by molar weight) member of La family, is succinctly outlined. Third, we describe genetic and nutritional factors that influence La production and export. A summary of the practical utility of the gained knowledge and future directions to study La biosynthesis conclude this mini-review.

Regulation of teicoplanin biosynthesis: refining the roles of tei cluster-situated regulatory genes.

Teicoplanin is a frontline glycopeptide antibiotic produced by Actinoplanes teichomyceticus. It is used to treat complicated cases of infection, including pediatric ones, caused by Gram-positive pathogens. There is a steady interest in elucidating the genetic mechanisms determining teicoplanin production, as they would help overproduce known teicoplanins and discover novel glycopeptides. Herein, we investigate the transcriptional organization of the tei biosynthetic gene cluster and the roles of the cluster-situated regulatory genes in controlling teicoplanin production and self-resistance in A. teichomyceticus. We demonstrate that the tei cluster is organized into nine polygenic and nine monogenic transcriptional units. Most of tei biosynthetic genes are subjected to StrR-like Tei15* control, which, in turn, appears to be regulated by LuxR-type Tei16*. Expression of the genes conferring teicoplanin self-resistance in A. teichomyceticus is not co-regulated with antibiotic production. The gene tei31*, coding for a putative DNA binding protein, is not expressed under teicoplanin producing conditions and is dispensable for antibiotic production. Finally, phylogenesis reconstruction of the glycopeptide cluster-encoded regulators reveals two main clades of StrR-like regulators. Tei15* and close orthologues form one of these clades; the second clade is composed by orthologues of Bbr and Dbv4, governing the biosynthesis of balhimycin and teicoplanin-like A40926, respectively. In addition, the LuxR-type Tei16* appears unrelated to the LuxR-like Dbv3, which is controlling A40926 biosynthesis. Our results shed new light on teicoplanin biosynthesis regulation and on the evolution of novel and old glycopeptide biosynthetic gene clusters.

Effect of "ribosome engineering" on the transcription level and production of S. albus indigenous secondary metabolites.

Significant resources are invested into efforts to improve the production yields of natural products from Actinobacteria, a well-recognized source of leads for several industries, most notably pharmaceutical one. Introduction of changes into genes for ribosomal protein S12 (rpsL) and/or 16S rRNA methylation (rsmG) is one of traditional approaches (referred to as ribosomal engineering) towards actinobacterial strain improvement. Yet, true potential of ribosome engineering remains unknown as it is currently coupled to empirical selection for aminoglycoside-resistance; rpsL mutations without such phenotypic expression could not be isolated. Here, we report a systematic and rational ribosome engineering approach to study the effect of a range of rpsL mutations on the production level of different biosynthetic gene clusters (BGC). The severe effect of diverse rpsL mutations together with deletion of rsmG engineered in Streptomyces albus has been revealed on the transcription level of several indigenous BGCs. The aforementioned mutations strongly impacted the transcription of indigenous BGCs, possibly because they alter the transcription of BGC-situated and global regulatory genes. The rsmG deletion with certain rpsL mutations can have a synergistic effect on the transcription level of indigenous BGCs. Our work thus provides the first streptomycete platform for rational engineering and study of virtually any nonlethal rpsL mutation. The tremendous effect of ribosome engineering on the transcription profile of the strains was reported for the first time. A library of described S. albus rpsL*/ΔrsmG strains represents a useful tool for overproducing known secondary metabolites and activating silent biosynthetic gene clusters in Actinobacteria.

Genome Engineering Approaches to Improve Nosokomycin A Production by Streptomyces ghanaensis B38.3.

Here we describe our efforts to improve the levels of phosphoglycolipid antibiotic nosokomycin A production by Streptomyces ghanaensis ATCC14672 via genome engineering approaches. Introduction of two extra copies of leucyl tRNA (UUA) gene bldA and one copy of moenomycin biosynthesis gene cluster led, on average, to threefold increase in nosokomycin A titers (up to 1.5 mg/L). Our results validate genome engineering approach as a viable strategy to improve moenomycin production.

Genomic Insights into Evolution of AdpA Family Master Regulators of Morphological Differentiation and Secondary Metabolism in Streptomyces.

The AdpA protein from a streptomycin producer Streptomyces griseus is a founding member of the AdpA family of pleiotropic regulators, known to be ubiquitously present in streptomycetes. Functional genomic approaches revealed a huge number of AdpA targets, leading to the claim that the AdpA regulon is the largest one in bacteria. The expression of adpA is limited at the level of translation of the rare leucyl UUA codon. All known properties of AdpA regulators were discovered on a few streptomycete strains. There are open questions about the true abundance and diversity of AdpA across actinobacterial taxa (and beyond) and about the possible evolutionary forces that shape the AdpA orthologous group in Streptomyces. Here we show that, with respect to the TTA codon, streptomycete adpA is more diverse than has been previously thought, as the genes differ in presence/position of this codon. Reciprocal best hits to AdpA can be found in many actinobacterial orders, with a domain organization resembling that of the prototypical AdpA, but other configurations also exist. Diversifying positive selection was detected within the DNA-binding (AraC) domain in adpA of Streptomyces origin, most likely affecting residues enabling AdpA to recognize a degenerate operator. Sequence coding for putative glutamine amidotransferase (GATase-1) domain also shows signs of positive selection. The two-domain organization of AdpA most likely arose from a fusion of genes encoding separate GATase-1 and AraC domains. Indeed, we show that the AraC domain retains a biological function in the absence of the GATase-1 part. We suggest that acquisition of the regulatory role by TTA codon is a relatively recent event in the evolution of AdpA, which coincided with the rise of the Streptomycetales clade and, at present, is under relaxed selective constraints. Further experimental scrutiny of our findings is invited, which should provide new insights into the evolution and prospects for engineering of an AdpA-centered regulatory network.

Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions.

Streptomyces cyanogenus S136 is the only known producer of landomycin A (LaA), one of the largest glycosylated angucycline antibiotics possessing strong antiproliferative properties. There is rising interest in elucidation of mechanisms of action of landomycins, which, in turn, requires access to large quantities of the pure compounds. Overproduction of LaA has been achieved in the past through manipulation of cluster-situated regulatory genes. However, other components of the LaA biosynthetic regulatory network remain unknown. To fill this gap, we elucidated the contribution of AdpA family pleiotropic regulators in landomycin production via expression of adpA genes of different origins in S. cyanogenus S136. Overexpression of the native S. cyanogenus S136 adpA ortholog had no effect on landomycin titers. In the same time, expression of several heterologous adpA genes led to significantly increased landomycin production under different cultivation conditions. Hence, heterologous adpA genes are a useful tool to enhance or activate landomycin production by S. cyanogenus. Our ongoing research effort is focused on identification of mutations that render S. cyanogenus AdpA nonfunctional.

Properties of Streptomyces albus J1074 mutant deficient in tRNALeuUAA gene bldA.

Streptomyces albus J1074 is one of the most popular and convenient hosts for heterologous expression of gene clusters directing the biosynthesis of various natural metabolic products, such as antibiotics. This fuels interest in elucidation of genetic mechanisms that may limit secondary metabolism in J1074. Here, we report the generation and initial study of J1074 mutant, deficient in gene bldA for tRNALeuUAA, the only tRNA capable of decoding rare leucyl TTA codon in Streptomyces. The bldA deletion in J1074 resulted in a highly conditional Bld phenotype, with depleted formation of aerial hyphae on certain solid media. In addition, bldA mutant of J1074 was unable to produce endogenous antibacterial compounds and two heterologous antibiotics, moenomycin and aranciamycin, whose biosynthesis is directed by TTA-containing genes. We have employed a new TTA codon-specific ß-galactosidase reporter system to provide genetic evidence that J1074 bldA mutant is impaired in translation of TTA. In addition, we have discussed the possible reasons for differences in the phenotypes of bldA mutants described here and in previous studies, providing knowledge to study bldA-based regulation of antibiotic biosynthesis.

A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.

Cell wall glycopolymers of Streptomyces albus, Streptomyces albidoflavus and Streptomyces pathocidini.

The cell wall glycopolymers of three strains of Streptomyces albus and the type strain of Streptomyces pathocidini were investigated. The structures of the glycopolymers were established using a combination of chemical and NMR spectroscopic methods. The cell wall of S. albus subsp. albus VKM Ac-35(T) was found to be comprised of three glycopolymers, viz. unsubstituted 1,5-poly(ribitol phosphate), 1,3-poly(glycerol phosphate) substituted with ß-D-glucopyranose, and the major polymer, a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid (Kdn)-teichulosonic acid: ß-D-Glcp-(1 â†’ 8)-α-Kdnp-(2[(→6)-ß-D-Glcp-(1 â†’ 8)-α-Kdnp-(2 â†’] n 6)-ß-D-Glcp-(1 â†’ 8)-ß-Kdnp-(2-OH, where n ≥ 3. The cell walls of 'S. albus' J1074 and 'S. albus' R1-100 were found to contain three glycopolymers of identical structures, viz. unsubstituted 1,3- and 2,3-poly(glycerol phosphates), and the major polymer, a Kdn-teichulosonic acid with an unusual structure that has not been previously described: ß-D-Galp-(1 â†’ 9)-α-Kdnp-(2[(→3)-ß-D-Galp-(1 â†’ 9)-α-Kdnp-(2 â†’] n 3)-ß-D-Galp-(1 â†’ 9)-ß-Kdnp-(2-OH, where n ~ 7-8. The cell wall of S. pathocidini (formerly S. albus subsp. pathocidicus) VKM Ac-598(T) was found to contain two glycopolymers, viz. 1,3-poly(glycerol phosphate) partially O-glycosylated with 2-acetamido-2-deoxy-α-D-glucopyranose and/or O-acylated with L-lysine, and a poly(diglycosyl 1-phosphate) of hitherto unknown structure: -6)-α-D-Glcp-(1 â†’ 6)-α-D-GlcpNAc-(1-P-.

The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression.

Analysis of the draft sequence of the genome of teicoplanin producer Actinoplanes teichomyceticus (NRRL-B16726) led to identification of several genes encoding AraC-family regulators that resemble AdpA, master regulator of transcription in Streptomyces. We elucidated possible regulatory functions of one of the identified genes, adpA19(at), most similar to archetypal adpA from model Streptomyces species, in a series of expression experiments. Introduction of adpA19 at under control of its own promoter on moderate copy number vector pKC1139 into NRRL-B16726 had no influence on antibiotic production and sporulation. Introduction of adpA19 at into Streptomyces coelicolor M145 and several S. ghanaensis strains had major influence on antibiotic production by these bacteria. Finally, adpA19 at expression in a set of soil actinomycete isolates led to induction of synthesis of antibiotic compounds. Our data point to pleiotropic regulatory role of adpA19(at), warranting its use as a tool to manipulate secondary metabolome of actinomycetes.

Insights into naturally minimised Streptomyces albus J1074 genome.

BACKGROUND: The Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery. RESULTS: To improve its capabilities for the heterologous expression of biosynthetic gene clusters, the complete genomic sequence of S. albus J1074 was obtained. With a size of 6,841,649 bp, coding for 5,832 genes, its genome is the smallest within the genus streptomycetes. Genome analysis revealed a strong tendency to reduce the number of genetic duplicates. The whole transcriptomes were sequenced at different time points to identify the early metabolic switch from the exponential to the stationary phase in S. albus J1074. CONCLUSIONS: S. albus J1074 carries the smallest genome among the completely sequenced species of the genus Streptomyces. The detailed genome and transcriptome analysis discloses its capability to serve as a premium host for the heterologous production of natural products. Moreover, the genome revealed 22 additional putative secondary metabolite gene clusters that reinforce the strain's potential for natural product synthesis.