UNLABELLED: ABCF-ATPases are increasingly recognized as translation factors that rescue stalled ribosomes when they encounter difficult mRNA templates or are stalled by antibiotics. The latter defines antibiotic resistance ABCF (ARE ABCF) proteins, known for their role in antibiotic resistance. However, in this study, we reveal a broader role of ARE ABCFs in antibiotic-responsive regulation. Using genetic, OMICs, and biochemical approaches, we showed that ARE ABCF proteins TiaA and Are5sc in Streptomyces coelicolor use their resistance functions to modulate specialized metabolism and proteosynthesis in response to lincosamide, streptogramin A, and pleuromutilin (LSAP) antibiotics. Although under LSAP exposure, either Are5sc or TiaA is essential for activating the biosynthesis of the redox-active antimicrobial actinorhodin, these proteins exhibit distinct functions at the proteome level, defined by their resistance profiles and temporally regulated expression. Are5sc facilitates early adaptive responses by modulating the WblC regulon across a broad range of LSAP concentrations, while TiaA is induced later, specifically at higher concentrations, where it suppresses antibiotic stress responses, particularly against pleuromutilins. TiaA function thus reflects the ecological context of LSAP antibiotics as pleuromutilins are produced by fungi, whereas lincosamides/streptogramins originate from actinomycetes. Our findings demonstrate that ARE ABCF proteins, through their resistance function, act as global regulators of translation, mirroring the roles of non-ARE ABCF proteins like EttA. This highlights their broader ecological and physiological significance, extending beyond their established role in antibiotic resistance. IMPORTANCE: Bacteria adapt to diverse stimuli mainly through transcriptional changes that regulate adaptive protein factors. Here, we show that responses to protein synthesis-inhibiting antibiotics are fine-tuned by antibiotic resistance ABCF proteins at the translational level, enabling bacteria to differentiate between antibiotic classes and concentrations for a tailored response. Additionally, we have demonstrated that these proteins can specialize in conferring high-level resistance to specific antibiotics. Given their prevalence in pathogenic bacteria, antibiotic resistance ABCF (ARE ABCF) proteins may play a crucial role in resistance development, particularly against new antibiotics targeting the ribosomal catalytic center, presenting a significant challenge for antimicrobial therapy.

• Keywords
• ABCF proteins, Streptomyces, antibiotic resistance, stress adaptation, stress response, translational control,
• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Drug Resistance, Bacterial * MeSH
• Bacterial Proteins * genetics   metabolism   MeSH
• Diterpenes pharmacology   MeSH
• Lincosamides pharmacology   MeSH
• Microbial Sensitivity Tests MeSH
• Pleuromutilins MeSH
• Polycyclic Compounds pharmacology   MeSH
• Protein Biosynthesis MeSH
• Gene Expression Regulation, Bacterial drug effects   MeSH
• Streptomyces coelicolor * drug effects   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• Bacterial Proteins * MeSH
• Diterpenes MeSH
• Lincosamides MeSH
• Pleuromutilins MeSH
• Polycyclic Compounds MeSH

Environmental microorganisms usually exhibit a high level of genomic plasticity and metabolic versatility that allow them to be well-adapted to diverse environmental challenges. This study used shotgun metagenomics to decipher the functional and metabolic attributes of an uncultured Paracoccus recovered from a polluted soil metagenome and determine whether the detected attributes are influenced by the nature of the polluted soil. Functional and metabolic attributes of the uncultured Paracoccus were elucidated via functional annotation of the open reading frames (ORFs) of its contig. Functional tools deployed for the analysis include KEGG, KEGG KofamKOALA, Clusters of Orthologous Groups of proteins (COG), Comprehensive Antibiotic Resistance Database (CARD), and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT V6) for antibiotic resistance genes, TnCentral for transposable element, Transporter Classification Database (TCDB) for transporter genes, and FunRich for gene enrichment analysis. Analyses revealed the preponderance of ABC transporter genes responsible for the transport of oligosaccharides (malK, msmX, msmK, lacK, smoK, aglK, togA, thuK, treV, msiK), monosaccharides (glcV, malK, rbsC, rbsA, araG, ytfR, mglA), amino acids (thiQ, ynjD, thiZ, glnQ, gluA, gltL, peb1C, artP, aotP, bgtA, artQ, artR), and several others. Also detected are transporter genes for inorganic/organic nutrients like phosphate/phosphonate, nitrate/nitrite/cyanate, sulfate/sulfonate, bicarbonate, and heavy metals such as nickel/cobalt, molybdate/tungstate, and iron, among others. Antibiotic resistance genes that mediate efflux, inactivation, and target protection were detected, while transposable elements carrying resistance phenotypes for antibiotics and heavy metals were also annotated. The findings from this study have established the resilience, adaptability, and survivability of the uncultured Paracoccus in the hydrocarbon-polluted soil.

• Keywords
• ABC transporters, Antibiotic resistance genes, Heavy metal resistance genes, Hydrocarbon-polluted soil, Transposable elements, Uncultured Paracoccus,
• MeSH
• ATP-Binding Cassette Transporters genetics   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Toxins * MeSH
• Clostridioides difficile * genetics   MeSH
• Metagenome MeSH
• Paracoccus * genetics   MeSH
• Soil chemistry   MeSH
• Metals, Heavy * MeSH
• DNA Transposable Elements MeSH
• Hydrocarbons MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Anti-Bacterial Agents MeSH
• Bacterial Toxins * MeSH
• Soil MeSH
• Metals, Heavy * MeSH
• DNA Transposable Elements MeSH
• Hydrocarbons MeSH

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.

• Keywords
• ABCF ATPase, Streptomyces, antibiotic biosynthesis, antibiotic resistance, antibiotic-mediated signaling, chemical communication, regulation of gene expression, ribosomal regulation, signal transduction, specialized metabolism,
• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Anti-Bacterial Agents biosynthesis   pharmacology   MeSH
• Drug Resistance, Bacterial MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Lincomycin biosynthesis   pharmacology   MeSH
• Methyltransferases MeSH
• Multigene Family MeSH
• Multidrug Resistance-Associated Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial drug effects   MeSH
• Ribosomes metabolism   MeSH
• Signal Transduction MeSH
• Streptomyces metabolism   MeSH
• Transcription Factors MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Lincomycin MeSH
• Methyltransferases MeSH
• Multidrug Resistance-Associated Proteins MeSH
• rRNA (adenosine-O-2'-)methyltransferase MeSH Browser
• Transcription Factors MeSH

Vga(A) protein variants confer different levels of resistance to lincosamides, streptogramin A, and pleuromutilins (LSAP) by displacing antibiotics from the ribosome. Here, we show that expression of vga(A) variants from Staphylococcus haemolyticus is regulated by cis-regulatory RNA in response to the LSAP antibiotics by the mechanism of ribosome-mediated attenuation. The specificity of induction depends on Vga(A)-mediated resistance rather than on the sequence of the riboregulator. Fine tuning between Vga(A) activity and its expression in response to the antibiotics may contribute to the selection of more potent Vga(A) variants because newly acquired mutation can be immediately phenotypically manifested.

• Keywords
• ABCF proteins, Staphylococcus haemolyticus, Vga(A), antibiotic resistance, clindamycin, lincosamides, pleuromutilins, regulation of gene expression, ribosome-mediated attenuation,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Lincosamides MeSH
• Macrolides MeSH
• Drug Resistance, Multiple, Bacterial * MeSH
• Ribosomes genetics   MeSH
• Streptogramin A * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Lincosamides MeSH
• Macrolides MeSH
• Streptogramin A * MeSH

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

• Keywords
• ABC ATPase, VmlR, antibiotic resistance, cryo-EM, ribosome,
• MeSH
• ATP-Binding Cassette Transporters chemistry   genetics   metabolism   MeSH
• Allosteric Regulation drug effects   genetics   MeSH
• Anti-Bacterial Agents chemistry   pharmacology   MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Drug Resistance, Bacterial * MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Ribosomes chemistry   genetics   metabolism   MeSH
• RNA, Transfer chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• RNA, Transfer MeSH