Heterologous expression reveals ancient properties of Tei3 - A 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 teichomyceticus—the producer of the last-resort-drug teicoplanin—has 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 ombining 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

Role of GntR family regulatory gene SCO1678 in gluconate metabolism in streptomyces coelicolor M145

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed

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 β-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

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNALeuUAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era

The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described

The use of the rare TTA codon in Streptomyces genes : significance of the codon context?

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)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

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

Hrab P, Rückert C, Busche T, et al. Complete genome sequence of Streptomyces cyanogenus S136, producer of anticancer angucycline landomycin A. 3 Biotech. 2021;11(6): 282 .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. © King Abdulaziz City for Science and Technology 2021

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

Yushchuk O, Ostash I, Vlasiuk I, et al. Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2018;102(19):8419-8428.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

Genomic insights into evolution of AdpA family master regulators of morphological differentiation and secondary metabolism in streptomyces

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)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