The resilience of Ukrainian scientists.

We have asked Ukrainian scientists how they have been able to persist in pursuing their research since the beginning of the full-scale invasion of Ukraine by the Russian Federation in February of 2022. We thank the scientists who were willing to share their thoughts and experiences; the views expressed are those of the contributors alone.

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.

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.

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.

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.

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.

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.

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.

Structural diversity, bioactivity, and biosynthesis of phosphoglycolipid family antibiotics: Recent advances.

Moenomycins, such as moenomycin A, are phosphoglycolipid specialized metabolites produced by a number of actinobacterial species. They are among the most potent antibacterial compounds known to date, which drew numerous studies directed at various aspects of the chemistry and biology of moenomycins. In this review, we outline the advances in moenomycin research over the last decade. We focus on biological aspects, highlighting the contribution of the novel methods of genomics and molecular biology to the deciphering of the biosynthesis and activity of moenomycins. Specifically, we describe the structural diversity of moenomycins as well as the underlying genomic variations in moenomycin biosynthetic gene clusters. We also describe the most recent data on the mechanism of action and assembly of complicated phosphoglycolipid scaffold. We conclude with the description of the genetic control of moenomycin production by Streptomyces bacteria and a brief outlook on future developments.

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.

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.

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.

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.

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.

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.

Genetic insights into the mechanism of teicoplanin self-resistance in Actinoplanes teichomyceticus.

Actinoplanes teichomyceticus NRRL B-16726 is the only known producer of the clinically important glycopeptide antibiotic teicoplanin. The producing strain is highly self-resistant to teicoplanin. Although the biosynthesis of teicoplanin has been investigated, much of our understanding of self-resistance in the producing strain is based on the extrapolation of existing data about glycopeptide resistance (mediated by the expression of vanRS-vanHAX genes) in other actinomycetes and cocci. The organization of the operons carrying putative van orthologues in A. teichomyceticus differs from known precedents, further adding up to the uncertainty about teicoplanin self-resistance mechanisms. Here, we determined operon structure of the teicoplanin resistance genes in A. teichomyceticus. Although Tei15* is necessary to activate teicoplanin biosynthetic genes, the expression of van orthologues was shown to be independent of Tei15*. We further showed that tei7 promoter driving the expression of vanHAX orthologues is dependent on Tei2 (VanR). Finally, we demonstrate the utility of the tei2 promoter as a new tool to achieve strong constitutive expression in A. teichomyceticus.

Development of a gene expression system for the uncommon actinomycete Actinoplanes rectilineatus NRRL B-16090.

The urgent need for discovering new bioactive metabolites prompts exploring novel actinobacterial taxa by developing appropriate tools for their genome mining and rational genetic engineering. One promising source of new bioactive natural products is the genus Actinoplanes, a home to filamentous sporangia-forming actinobacteria producing many important specialized metabolites such as teicoplanin, ramoplanin, and acarbose. Here we describe the development of a gene expression system for a new Actinoplanes species, A. rectilineatus (NRRL B-16090), which is a potential producer of moenomycin-like antibiotics. We have determined the optimal conditions for spore formation in A. rectilineatus and a plasmid transfer procedure for its engineering via intergeneric E. coli-A. rectilineatus conjugation. The φC31- and pSG5-based vectors were successfully transferred into A. rectilineatus, but φBT1- and VWB-based vectors were not transferable. Finally, using the glucuronidase reporter system, we assessed the strength of several heterologous promoters for gene expression in A. rectilineatus.

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.

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.

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.