In natural environments, antibiotics are important means of interspecies competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production; however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis, we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize antibiotic production within the population. The entire cascade is encoded in the lincomycin biosynthetic gene cluster (BGC) and consists of three lincomycin resistance proteins in addition to the transcriptional regulator LmbU: a lincomycin transporter (LmrA), a 23S rRNA methyltransferase (LmrB), both of which confer high resistance, and an ATP-binding cassette family F (ABCF) ATPase, LmrC, which confers only moderate resistance but is essential for antibiotic-induced signal transduction. Specifically, antibiotic sensing occurs via ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, ATPase activity of the ribosome-associated LmrC triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB, which reduces the amount of ribosome-bound antibiotic and thus fine-tunes the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many ribosome-targeting antibiotic BGCs encode an ABCF protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of the eight coproduced ABCF proteins of S. lincolnensis are clindamycin responsive, suggesting that the ABCF-mediated antibiotic signaling may be a widely utilized tool for chemical communication. IMPORTANCE Resistance proteins are perceived as mechanisms protecting bacteria from the inhibitory effect of their produced antibiotics or antibiotics from competitors. Here, we report that antibiotic resistance proteins regulate lincomycin biosynthesis in response to subinhibitory concentrations of antibiotics. In particular, we show the dual character of the ABCF ATPase LmrC, which confers antibiotic resistance and simultaneously transduces a signal from ribosome-bound antibiotics to gene expression, where the 5' untranslated sequence upstream of its encoding gene functions as a primary antibiotic sensor. ABCF-mediated antibiotic signaling can in principle function not only in the induction of antibiotic biosynthesis but also in selective gene expression in response to any small molecules targeting the 50S ribosomal subunit, including clinically important antibiotics, to mediate intercellular antibiotic signaling and stress response induction. Moreover, the resistance-regulatory function of LmrC presented here for the first time unifies functionally inconsistent ABCF family members involving antibiotic resistance proteins and translational regulators.

• Klíčová slova
• ABCF ATPase, Streptomyces, antibiotic biosynthesis, antibiotic resistance, antibiotic-mediated signaling, chemical communication, regulation of gene expression, ribosomal regulation, signal transduction, specialized metabolism,
• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• antibakteriální látky biosyntéza   farmakologie   MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• linkomycin biosyntéza   farmakologie   MeSH
• methyltransferasy MeSH
• multigenová rodina MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům genetika   metabolismus   MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• ribozomy metabolismus   MeSH
• signální transdukce MeSH
• Streptomyces metabolismus   MeSH
• transkripční faktory MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• linkomycin MeSH
• methyltransferasy MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům MeSH
• rRNA (adenosine-O-2'-)methyltransferase MeSH Prohlížeč
• transkripční faktory MeSH

HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální RNA genetika   MeSH
• chromatinová imunoprecipitace MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• exprese genu MeSH
• geny rRNA MeSH
• kinetika MeSH
• modely genetické MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií MeSH
• regulon * MeSH
• RNA transferová genetika   MeSH
• sekvenční analýza DNA MeSH
• sigma faktor metabolismus   MeSH
• Streptomyces coelicolor genetika   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• DNA bakterií MeSH
• DNA vazebné proteiny MeSH
• HrdB protein, Streptomyces MeSH Prohlížeč
• RNA transferová MeSH
• sigma faktor MeSH

Low molecular weight signaling compounds (LMWC) are important players in regulating various aspects of Streptomyces biology. Their exact roles in certain strain will ultimately depend on overall configuration of regulatory network and thus cannot be predicted on basis of in silico studies. Here, we explored S. ghanaensis gene SSFG_07725 (afsAgh) presumably involved in initial steps of formation of γ-butyrolactone LMWC. Disruption of afsAgh impaired aerial mycelium formation and increased the transcription of pleiotropic regulatory gene adpAgh, whereas level of moenomycin production remained virtually unaffected. We provide evidence that morphogenetic deficiency of afsAgh-minus mutant was caused by inability to produce diffusible LMWC. Possible links between γ-butyrolactone signaling and various aspects of S. ghanaensis biology are discussed.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• gama-butyrolakton metabolismus   MeSH
• mutace MeSH
• oligosacharidy metabolismus   MeSH
• Streptomyces enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• gama-butyrolakton MeSH
• moenomycin MeSH Prohlížeč
• oligosacharidy MeSH

In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

• MeSH
• cystein metabolismus   MeSH
• glykopeptidy metabolismus   MeSH
• inositol metabolismus   MeSH
• linkomycin biosyntéza   MeSH
• linkosamidy biosyntéza   MeSH
• peptidsynthasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celesticetin A MeSH Prohlížeč
• cystein MeSH
• glykopeptidy MeSH
• inositol MeSH
• linkomycin MeSH
• linkosamidy MeSH
• mycothiol MeSH Prohlížeč
• peptidsynthasy MeSH

The antibiotic L-155,175, a potent antiparasitic and antifungal compound, has an unusual structure involving 16-membered macrolides that contain a tetrahydropyran ring connected through a three-carbon linker chain. To identify the biosynthetic gene cluster for L-155,175, a genomic DNA library of Streptomyces hygroscopicus ATCC31955 was constructed and screened with a degenerate primer set designed from a conserved region of the ketosynthase (KS) domain. Sequence analysis of a fosmid clone, pEY1D8 (34 kb), revealed multiple open reading frames (ORFs) encoding type I polyketide synthase (PKS). To determine whether the cloned genes are involved in L-155,175 biosynthesis, a deletion mutant (1D8m) was generated by homologous recombination, in which the gene encoding the KS domain was substituted with an apramycin-resistance gene by PCR-targeted Streptomyces gene replacement. LC-MS analysis showed that L-155,175 production was completely abolished in the 1D8m strain, thereby proving that the cloned gene is responsible for L-155,175 biosynthesis. The sequencing of two other fosmid clones (pEY8B10 and pEY1C9) harboring overlapping sequences from pEY1D8 revealed a 60-kb DNA segment encoding six ORFs for type I PKS harboring 12 modules. The domain organization of the PKS modules encoded by PKS exactly matched the structure of L-155,175. This is the first report on the gene cluster involved in the biosynthesis of L-155,175.

• MeSH
• antiinfekční látky metabolismus   MeSH
• biosyntetické dráhy genetika   MeSH
• DNA bakterií chemie   genetika   MeSH
• genová knihovna MeSH
• genový knockout MeSH
• makrolidy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina * MeSH
• otevřené čtecí rámce MeSH
• polyketidy metabolismus   MeSH
• sekvenční analýza DNA MeSH
• Streptomyces genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiinfekční látky MeSH
• DNA bakterií MeSH
• hygrolidin K2 MeSH Prohlížeč
• makrolidy MeSH
• polyketidy MeSH

The polyketide gene cluster aur1 is responsible for the production of the antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is low and strictly regulated by two regulators, Aur1P and Aur1R. To improve auricin yield, we genetically manipulated S. aureofaciens CCM 3239 strain to overcome this strict regulation. A regulatory region including aur1R, aur1P, aur1O and the target biosynthetic aur1Ap promoter were replaced by the strong constitutive ermEp* promoter. However, auricin production was decreased in such a genetically manipulated strain. In the second strategy we placed the aur1P gene for auricin pathway-specific activator under the control of the ermEp* promoter. The resulting strain has been shown to produce 2.8-fold higher amount of auricin compared with the WT strain.

• MeSH
• antibakteriální látky biosyntéza   MeSH
• DNA bakterií genetika   metabolismus   MeSH
• makrolidy metabolismus   MeSH
• multigenová rodina MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií MeSH
• restrikční mapování MeSH
• Streptomyces aureofaciens genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• auricin MeSH Prohlížeč
• DNA bakterií MeSH
• makrolidy MeSH

An incomplete oligoketide (PK; 'polyketide') gene cluster, aur1, responsible for the production of an angucycline-like antibiotic auricin was identified in Streptomyces aureofaciens CCM 3239. A region downstream of the aur1 was cloned and sequenced, revealing 28 new genes encoding putative protein products involved in deoxysugar biosynthesis and other putative PK-related biosynthetic functions. In addition, a gene, bpsA, encoding a protein similar to non-ribosomal peptide synthetases (NRPSs) was identified in this region. A deduced protein product of the gene showed the highest similarity to NRPSs IndC from Erwinia chrysanthemi and BpsA from Streptomyces lavendulae, both involved in the biosynthesis of a blue pigment indigoidine. S. aureofaciens CCM 3239 was found to produce an extracellular blue pigment with identical properties as indigoidine. A deletion mutant of bpsA in S. aureofaciens CCM 3239 failed to produce the blue pigment. In addition, the deletion of bpsA had a positive effect on auricin production. The results indicate the involvement of the bpsA gene in biosynthesis of the indigoidine blue pigment in S. aureofaciens CCM 3239.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biologické pigmenty biosyntéza   MeSH
• molekulární sekvence - údaje MeSH
• piperidony metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Streptomyces aureofaciens genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• biologické pigmenty MeSH
• indigoidine MeSH Prohlížeč
• piperidony MeSH

The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-L-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4'-butyl-4'-depropyllincomycin and 4'-pentyl-4'-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4'-pentyl-4'-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

• MeSH
• antibakteriální látky chemie   metabolismus   farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• lidé MeSH
• linkomycin analogy a deriváty   chemie   metabolismus   farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• prolin analogy a deriváty   metabolismus   MeSH
• stafylokokové infekce mikrobiologie   MeSH
• Staphylococcus účinky léků   MeSH
• Streptomyces genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• linkomycin MeSH
• prolin MeSH