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.

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.

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.

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.

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.

Mutations within gene XNR_2147 for TetR-like protein enhance lincomycin resistance and endogenous specialized metabolism of Streptomyces albus J1074.

Streptomyces albus J1074 is one of the most popular heterologous expression platforms among streptomycetes. Identification of new genes and mutations that influence specialized metabolism in this species is therefore of great applied interest. Here, we describe S. albus KO-1304 that was isolated as a spontaneous lincomycin-resistant variant of double rpsLR94G rsmGR15SG40E mutant KO-1295. Besides altered antibiotic resistance profile, KO-1304 exhibited increased antibiotic activity as compared to its parental strains. KO-1304 genome sequencing revealed mutations within gene XNR_2147 encoding putative TetR-like protein. Gene XNR_2146 for efflux protein is the most likely target of repressing action of Xnr_2147. Our data agree with the scenario where lincomycin resistance phenotype of KO-1304 arose from inability of mutated Xnr_2147 protein to repress XNR_2146. Introduction of additional copy of XNR_2146 into wild type strain increased antibiotic activity of the latter, attesting to the practical value of transporter genes for strain improvement.

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074.

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the ß-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites.

The carbohydrate tail of landomycin A is responsible for its interaction with the repressor protein LanK.

Landomycin A (LaA) is the largest member of the landomycin group of angucyclic polyketides. Its unusual structure and strong anticancer properties have attracted great interest from chemists and biologists alike. This, in particular, has led to a detailed picture of LaA biosynthesis in Streptomyces cyanogenus S136, the only known LaA producer. LanK is a TetR family repressor protein that limits the export of landomycins from S136 cells until significant amounts of the final product, LaA, have accumulated. Landomycins carrying three or more carbohydrate units in their glycosidic chain are effector molecules for LanK. Yet, the exact mechanism that LanK uses to distinguish the final product, LaA, from intermediate landomycins and sense accumulation of LaA was not known. Here, we report crystal structures of LanK, alone and in complex with LaA, and bioassays of LanK's interaction with synthetic carbohydrate chains of LaA (hexasaccharide) and LaE (trisaccharide). Our data collectively suggest that the carbohydrate moieties are the sole determinants of the interaction of the landomycins with LanK, triggering the latter's dissociation from the lanK-lanJ intergenic region via structure conversion of the helices in the C-terminal ligand-binding domain. Analysis of the available literature suggests that LanK represents an unprecedented type of TetR family repressor that recognises the carbohydrate portion of a natural product, and not an aglycon, as it is the case, for example, with the SimR repressor involved in simocyclinone biosynthesis.

Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production.

Here, we report the identification and functional characterization of the Streptomyces globisporus 1912 gene lndYR, which encodes a GntR-like regulator of the YtrA subfamily. Disruption of lndYR arrested sporulation and antibiotic production in S. globisporus. The results of in vivo and in vitro studies revealed that the ABC transporter genes lndW-lndW2 are targets of LndYR repressive action. In Streptomyces coelicolor M145, lndYR overexpression caused a significant increase in the amount of extracellular actinorhodin. We suggest that lndYR controls the transcription of transport system genes in response to an as-yet-unidentified signal. Features that distinguish lndYR-based regulation from other known regulators are discussed.

Complete genome sequence of Streptomyces cyanogenus S136, producer of anticancer angucycline landomycin A.

Streptomyces cyanogenus S136 is the only known producer of landomycin A (LaA), one of the founding members of angucycline family of aromatic polyketides. LaA displays potent anticancer activities which has made this natural product a target of numerous chemical and cell biological studies. Little is known about the potential of S136 strain to produce other secondary metabolites. Here we report complete genome sequence of LaA producer and how we used this sequence to evaluate for this species its phylogenetic position and diversity of gene clusters for natural product biosynthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02834-4.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Genetic analysis of Streptomyces albus J1074 mia mutants suggests complex relationships between post-transcriptional tRNAXXA modifications and physiological traits.

Proteins MiaA and MiaB catalyze sequential isopentenylation and methylthiolation, respectively, of adenosine residue in 37th position of tRNAXXA. The mia mutations were recently shown by us to affect secondary metabolism and morphology of Streptomyces. However, it remained unknown as to whether both or one of the aforementioned modifications is critical for colony development and antibiotic production. Here, we addressed this issue through analysis of Streptomyces albus J1074 strains carrying double miaAmiaB knockout or extra copy of miaB gene. The double mutant differed from wild-type and miaA-minus strains in severity of morphological defects, growth dynamics, and secondary metabolism. Introduction of extra copy of miaB gene into miaA mutant restored aerial mycelium formation to the latter on certain solid media. Hence, miaB gene might be involved in tRNA thiomethylation in the absence of miaA; either MiaA- or MiaB-mediated modification appears to be enough to support normal metabolic and morphological processes in Streptomyces.

Complete characterization of the seventeen step moenomycin biosynthetic pathway.

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

Coordination of export and glycosylation of landomycins in Streptomyces cyanogenus S136.

In Streptomyces cyanogenus S136 gene cluster for biosynthesis of polyglycosylated angucycline landomycin A (LaA), a divergently oriented gene pair for a TetR-family regulator (lanK) and an efflux protein (lanJ) is located, whose functions remained obscure. Overexpression and disruption studies showed that lanK and lanJ genes control LaA resistance. Also, a constitutive lanK overexpression led to predominant accumulation of LaA precursors bearing shorter glycoside chains. These data as well as the results of in vitro and in vivo assays of LanK activity are consistent with the idea that LanK represses lanJ and some downstream genes involved in conversion of landomycin D (a disaccharide LaA precursor) into LaA. LaA and some of its precursors accumulate in the producing cell and relieve repression by LanK, thus amplifying the biosynthesis and export of landomycins with long glycoside chains. Therefore, the main biological role of LanK appears to be the inhibition of premature extrusion of early LaA precursors from the cells, which in turn creates the optimal conditions for accumulation of LaA as the major landomycin in S. cyanogenus S136.

An ABC transporter encoding gene lndW confers resistance to landomycin E.

Streptomyces globisporus 1912 produces a polyketide antibiotic landomycin E (LaE), which possesses anticancer activity. A 1.8 kb DNA fragment at the end of landomycin E biosynthetic gene cluster was sequenced. DNA sequence analysis of this fragment identified one complete open reading frame, designated lndW. The deduced sequence of lndW gene product revealed significant similarity to the ATP-binding domains of the ABC (ATP-binding protein cassette) superfamily of transport-related proteins. Knockout of lndW had no significant effect on resistance to LaE and its production. The expression of lndW in S. globisporus 1912 was proven via transcriptional fusion of lndW promoter to EGFP (enhanced green fluorescent protein). Overexpression of lndW in S. lividans TK24 conferred resistance to LaE. The mechanism of lndW function in LaE biosynthesis is discussed.