Beyond Self-Resistance: ABCF ATPase LmrC Is a Signal-Transducing Component of an Antibiotic-Driven Signaling Cascade Accelerating the Onset of Lincomycin Biosynthesis.

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.

R/G Value-A Numeric Index of Individual Periodontal Health and Oral Microbiome Dynamics.

The dysbiosis of oral microbiome (OM) precedes the clinical signs of periodontal disease. Its simple measure thus could indicate individuals at risk of periodontitis development; however, such a tool is still missing. Up to now, numerous microbial taxa were associated with periodontal health or periodontitis. The outputs of most studies could, nevertheless, be slightly biased from following two reasons: First, the healthy group is often characterized only by the absence of the disease, but the individuals could already suffer from dysbiosis without any visible signs. Second, the healthy/diseased OM characteristics are frequently determined based on average data obtained for whole groups of periodontally healthy persons versus patients. Especially in smaller sets of tested individuals the typical individual variability can thus complicate the unambiguous assignment of oral taxa to respective state of health. In this work the taxonomic composition of OM was evaluated for 20 periodontally healthy individuals and 15 patients with chronic periodontitis. The narrowed selection set of the most diseased patients (confirmed by clinical parameters) and the most distant group of healthy individuals with the lowest probability of dysbiosis was determined by clustering analysis and used for identification of marker taxa. Based on their representation in each individual oral cavity we proposed the numeric index of periodontal health called R/G value. Its diagnostic potential was further confirmed using independent set of 20 periodontally healthy individuals and 20 patients with periodontitis with 95 percent of samples assigned correctly. We also assessed the individual temporal OM dynamics in periodontal health and we compared it to periodontitis. We revealed that the taxonomic composition of the system changes dynamically but generally it ranges within values typical for periodontal health or transient state, but far from values typical for periodontitis. R/G value tool, formulated from individually evaluated data, allowed us to arrange individual OMs into a continuous series, instead of two distinct groups, thus mimicking the gradual transformation of a virtual person from periodontal health to disease. The application of R/G value index thus represents a very promising diagnostic tool for early prediction of persons at risk of developing periodontal disease.

Ribosome-Mediated Attenuation of vga(A) Expression Is Shaped by the Antibiotic Resistance Specificity of Vga(A) Protein Variants.

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.

Lincosamide synthetase--a unique condensation system combining elements of nonribosomal peptide synthetase and mycothiol metabolism.

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.

Sequence analysis of porothramycin biosynthetic gene cluster.

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.

Adaptation of an L-proline adenylation domain to use 4-propyl-L-proline in the evolution of lincosamide biosynthesis.

Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.

High-throughput quantification of lincomycin traces in fermentation broth of genetically modified Streptomyces spp. Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography with UV detection.

A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.