A synthetic antibiotic class overcoming bacterial multidrug resistance.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.

Mutant noxy8 exposes functional specificities between the chloroplast chaperones CLPC1 and CLPC2 in the response to organelle stress and plant defence.

Chloroplast function is essential for growth, development, and plant adaptation to stress. Organelle stress and plant defence responses were examined here using noxy8 (nonresponding to oxylipins 8) from a series of Arabidopsis mutants. The noxy8 mutation was located at the CLPC2 gene, encoding a chloroplast chaperone of the protease complex CLP. Although its CLPC1 paralogue is considered to generate redundancy, our data reveal significant differences distinguishing CLPC2 and CLPC1 functions. As such, clpc1 mutants displayed a major defect in housekeeping chloroplast proteostasis, leading to a pronounced reduction in growth and pigment levels, enhanced accumulation of chloroplast and cytosol chaperones, and resistance to fosmidomycin. Conversely, clpc2 mutants showed severe susceptibility to lincomycin inhibition of chloroplast translation and resistance to Antimycin A inhibition of mitochondrial respiration. In the response to Pseudomonas syringae pv. tomato, clpc2 but not clpc1 mutants were resistant to bacterial infection, showing higher salicylic acid levels, defence gene expression and 9-LOX pathway activation. Our findings suggest CLPC2 and CLPC1 functional specificity, with a preferential involvement of CLPC1 in housekeeping processes and of CLPC2 in stress responses.

Baicalin restore intestinal damage after early-life antibiotic therapy: the role of the MAPK signaling pathway.

Antibiotic related intestinal injury in early life affects subsequent health and susceptibility. Here, we employed weaned piglets as a model to investigate the protective effects of baicalin against early-life antibiotic exposure-induced microbial dysbiosis. Piglets exposed to lincomycin showed a marked reduction in body weight (p < 0.05) and deterioration of jejunum intestinal morphology, alongside an increase in antibiotic-resistant bacteria such as Staphylococcus, Dolosicoccus, Escherichia-Shigella, and Raoultella. In contrast, baicalin treatment resulted in body weights, intestinal morphology, and microbial profiles that closely resembled those of the control group (p > 0.05), with a significant increase in norank_f_Muribaculaceae and Prevotellaceae_NK3B31_group colonization compared with lincomycin group (p < 0.05). Further analysis through fecal microbial transplantation into mice revealed that lincomycin exposure led to significant alterations in intestinal morphology and microbial composition, notably increasing harmful microbes and decreasing beneficial ones such as norank_Muribaculaceae and Akkermansia (p < 0.05). This shift was associated with an increase in harmful metabolites and disruption of the calcium signaling pathway gene expression. Conversely, baicalin supplementation not only counteracted these effects but also enhanced beneficial metabolites and regulated genes within the MAPK signaling pathway (MAP3K11, MAP4K2, MAPK7, MAPK13) and calcium channel proteins (ORA13, CACNA1S, CACNA1F and CACNG8), suggesting a mechanism through which baicalin mitigates antibiotic-induced intestinal and microbial disturbances. These findings highlight baicalin's potential as a plant extract-based intervention for preventing antibiotic-related intestinal injury and offer new targets for therapeutic strategies.

Effects of the pleiotropic regulator DasR on lincomycin production in Streptomyces lincolnensis.

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.

Potassium alleviates over-reduction of the photosynthetic electron transport chain and helps to maintain photosynthetic function under salt-stress.

Potassium ions enhance photosynthetic tolerance to salt stress. We hypothesized that potassium ions, by minimizing the trans-thylakoid proton diffusion potential difference, can alleviate over-reduction of the photosynthetic electron transport chain and maintain the functionality of the photosynthetic apparatus. This study investigated the effects of exogenous potassium on the transcription level and activity of proteins related to the photosynthetic electron-transport chain of tobacco seedlings under salt stress. Salt stress retarded the growth of seedlings and caused an outflow of potassium ions from the chloroplast. It also lowered qP (indicator of the oxidation state of QA , the primary quinone electron acceptor in Photosystem II (PSII) and YPSII (average photochemical yield of PSII in the light-adapted state) while increasing YNO+NF (nonregulatory energy dissipation in functional and nonfunctional PSII), accompanied by decreased expression of most light-harvesting, energy-transduction, and electron-transport genes. However, exogenous potassium prevented these effects due to NaCl. Interestingly, lincomycin (an inhibitor of the synthesis of chloroplast-encoded proteins in PSII) significantly diminished the alleviation effect of exogenous potassium on salt stress. We attribute the comprehensive NaCl-induced downregulation of transcription and photosynthetic activities to retrograde signaling induced by reactive oxygen species. There probably exist at least two types of retrograde signaling induced by reactive oxygen species, distinguished by their sensitivity to lincomycin. Exogenous potassium appears to exert its primary effect by ameliorating the trans-thylakoid proton diffusion potential difference via a potassium channel, thereby accelerating ATP synthesis and carbon assimilation, alleviating over-reduction of the photosynthetic electron transport chain, and maintaining the functionality of photosynthetic proteins.

Characterization of a TetR-type positive regulator AtrA for lincomycin production in Streptomyces lincolnensis.

AtrA belongs to the TetR family and has been well characterized for its roles in antibiotic biosynthesis regulation. Here, we identified an AtrA homolog (AtrA-lin) in Streptomyces lincolnensis. Disruption of atrA-lin resulted in reduced lincomycin production, whereas the complement restored the lincomycin production level to that of the wild-type. In addition, atrA-lin disruption did not affect cell growth and morphological differentiation. Furthermore, atrA-lin disruption hindered the transcription of regulatory gene lmbU, structural genes lmbA and lmbW inside the lincomycin biosynthesis gene cluster, and 2 other regulatory genes, adpA and bldA. Completement of atrA-lin restored the transcription of these genes to varying degrees. Notably, we found that AtrA-lin directly binds to the promoter region of lmbU. Collectively, AtrA-lin positively modulated lincomycin production via both pathway-specific and global regulators. This study offers further insights into the functional diversity of AtrA homologs and the mechanism of lincomycin biosynthesis regulation.

Penicillin and amikacin mixture has bactericidal activity equivalent to gentamicin, tylosin, lincomycin and spectinomycin mixture in equine frozen semen.

Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, a bioequivalence study was performed to assess the bactericidal activity of GTLS and PA in equine frozen semen. Nine mature, healthy stallions were used in the study. Split ejaculates were processed using media without antibiotics (Control) or with different antibiotics. For the GTLS group, centrifugation medium and freezing extender were prepared with gentamicin 250 µg/ml, tylosin 50 µg/ml, lincomycin 150 µg/ml and spectinomycin 300 µg/ml. For the PA group, the centrifugation medium was prepared with potassium penicillin G (PPG) 1200 units/ml and the freezing extender was prepared with PPG 1200 units/ml and amikacin 500 µg/ml. Semen processed in extenders without antibiotics had higher (p < .005) bacterial loads throughout all cryopreservation processing steps than semen samples processed using antibiotics. There were no differences in semen bacterial load after centrifugation, 15 and 30 min after final extension, and after thawing between GTLS and PA groups, but PA had faster (p < .05) kill-time kinetics than GTLS. Only minor differences in sperm kinetic parameters were observed among groups. In conclusion, this study demonstrated bioequivalence between GTLS and PA in mitigating end-point bacterial loads. Prudent concentrations of the antibiotic mixtures evaluated in this study can be considered both effective and sperm-safe for equine frozen semen.

In vitro susceptibility of Mycoplasma iowae isolates to antimicrobial agents.

ABSTRACTMycoplasma iowae, a potential re-emerging avian pathogen mainly affecting turkeys, has been reported from many parts of the world. Poor hatchability, embryonic death, joint and skeletal abnormalities, poor ossification, runting-stunting, poor feathering and airsacculitis may be observed in infected flocks. The reduction of the severity of clinical signs and short-term control of M. iowae are performed by antibiotic treatment. However, M. iowae develops resistance more rapidly and is considered to be more resistant to antimicrobials than other avian pathogenic mycoplasmas. The aim of the present study was to determine the in vitro susceptibility of 101 M. iowae isolates and strains to ten clinically important antimicrobial agents, and to analyse and compare the susceptibility patterns of isolates of various origins and from a wide time-period. The examined reference strains showed high susceptibility to all antimicrobials except for spectinomycin. Low concentrations of tiamulin, florfenicol and oxytetracycline inhibited the growth of the clinical isolates. Nevertheless, slow tendency of increasing minimum inhibitory concentration (MIC) values was observed over time in the case of the above mentioned agents, while MIC values of enrofloxacin showed relatively rapid changes. Spiramycin, erythromycin, tilmicosin, tylosin, lincomycin and spectinomycin did not inhibit the bacterial growth in most of the cases. Isolates originating from captive game birds showed similar susceptibility profiles to isolates from industrial turkey hosts. The widely detected low susceptibility of M. iowae isolates to macrolides, lincomycin and spectinomycin, and the increase of MIC values of frequently used antimicrobials against this pathogen, emphasize the importance of targeted antibiotic therapy.RESEARCH HIGHLIGHTSAntimicrobial susceptibilities of 101 Mycoplasma iowae isolates were determined.Minimum inhibitory concentrations were determined by broth micro-dilution method.Tiamulin, oxytetracycline and florfenicol showed low MIC values.Isolates rapidly adapted to antimicrobial pressure.

Residual concentrations of antimicrobial growth promoters in poultry litter favour plasmid conjugation among Escherichia coli.

Considering that plasmid conjugation is a major driver for the dissemination of antimicrobial resistance in bacteria, this study aimed to investigate the effects of residual concentrations of antimicrobial growth promoters (AGPs) in poultry litter on the frequencies of IncFII-FIB plasmid conjugation among Escherichia coli organisms. A 2 × 5 factorial trial was performed in vitro, using two types of litter materials (sugarcane bagasse and wood shavings) and five treatments of litter: non-treated (CON), herbal alkaloid sanguinarine (SANG), AGPs monensin (MON), lincomycin (LCM) and virginiamycin (VIR). E. coli H2332 and E. coli J62 were used as donor and recipient strains, respectively. The presence of residues of monensin, lincomycin and virginiamycin increased the frequency of plasmid conjugation among E. coli in both types of litter materials. On the contrary, sanguinarine significantly reduced the frequency of conjugation among E. coli in sugarcane bagasse litter. The conjugation frequencies were significantly higher in wood shavings compared with sugarcane bagasse only in the presence of AGPs. Considering that the presence of AGPs in the litter can increase the conjugation of IncFII-FIB plasmids carrying antimicrobial resistance genes, the real impact of this phenomenon on the dissemination of antimicrobial resistant bacteria in the poultry production chain must be investigated.

Baicalin Alleviates Short-Term Lincomycin-Induced Intestinal and Liver Injury and Inflammation in Infant Mice.

The adverse effects of short-term megadose of antibiotics exposure on the gastrointestinal and liver tissue reactions in young children have been reported. Antibiotic-induced intestinal and liver reactions are usually unpredictable and present a poorly understood pathogenesis. It is, therefore, necessary to develop strategies for reducing the adverse effects of antibiotics. Studies on the harm and rescue measures of antibiotics from the perspective of the gut-liver system are lacking. Here, we demonstrate that lincomycin exposure reduced body weight, disrupted the composition of gut microbiota and intestinal morphology, triggered immune-mediated injury and inflammation, caused liver dysfunction, and affected lipid metabolism. However, baicalin administration attenuated the lincomycin-induced changes. Transcriptome analysis showed that baicalin improved immunity in mice, as evidenced by the decreased levels of intestinal inflammatory cytokines and expression of genes that regulate Th1, Th2, and Th17 cell differentiation, and inhibited mucin type O-glycan biosynthesis pathways. In addition, baicalin improved liver function by upregulating the expression of genes involved in bile acid secretion and lipid degradation, and downregulating genes involved in lipid synthesis in lincomycin-treated mice. Bile acids can regulate intestinal immunity and strengthen hepatoenteric circulation. In addition, baicalin also improved anti-inflammatory bacteria abundance (Blautia and Coprobacillus) and reduced pathogenic bacteria abundance (Proteobacteria, Klebsiella, and Citrobacter) in lincomycin-treated mice. Thus, baicalin can ameliorate antibiotic-induced injury and its associated complications such as liver disease.

Hemolytic Activity, Cytotoxicity, and Antimicrobial Effects of Silver Nanoparticles Conjugated with Lincomycin or Cefazolin.

The overuse of antibiotics has led to the emergence of resistant bacteria. A good alternative is silver nanoparticles, which have antibacterial activity against Gram-negative and Gram-positive bacteria, including multidrug-resistant strains. Their combination with already known antibiotics has a synergistic effect. In this work, we studied the synthesis of conjugates of silver nanoparticles with two antibiotics, lincomycin and cefazolin. Albumin and glutathione were used as spacer shells with functional groups. The physicochemical properties of the obtained conjugates, their cytotoxicity and synergism of antimicrobial activity were studied. The 50% antimicrobial activity of the obtained samples was shown, which allows them to be recommended for use as topical drug preparations.

Antibiogram of microorganisms isolated from fresh and frozen semen of crossbred frieswal bulls.

BACKGROUND: The bacterial contaminants in the semen are a major concern for most of the semen production laboratories because they adversely affect the semen quality. During sperm cryopreservation, the inclusion of antimicrobials in extenders may help to minimize bacterial growth. However, due to bacterial resistance to commonly used antimicrobials, they cannot fully assure microbiological safety to the frozen semen. OBJECTIVE: To estimate the microbial load and antibiogram of microorganisms isolated from the fresh and frozen bull semen. MATERIALS AND METHODS: The bacterial load was estimated in fresh and frozen semen samples of crossbred Frieswal bulls by the pour plate method. Microorganisms were identified as Gram positive and Gram negative by Gram staining. The representative bacterial colonies were streaked onto different specific media which were further confirmed by biochemical tests. Bacterial isolates were subjected to in vitro antibiotic sensitivity test. RESULTS: The average microbial load of fresh and frozen semen samples was found to be 8397.4 ± 524.3 cfu per mL and 680.9 ± 105.4 cfuper mL, respectively. Microorganisms belonging to Staphylococcus aureus, Staphylococcus epidermidis, Proteus, Klebsiella, Bacillus cereus, Bacillus subtilis, Actinomyces, E. coli, Rhodococcus, Neisseria and Micrococcus were identified in the semen samples. The antibiotic sensitivity testing of the bacterial isolates revealed that benzyl penicillin was found to be the least effective against the isolated organisms while gentamicin and spectinomycin were found to be most effective among the antibiotics used. Lincomycin, tylosin and streptomycin showed moderate efficacy against the bacterial isolates. CONCLUSION: Gentamicin, tylosin, lincomycin, and spectinomycin (GTLS) antibiotic combination is more effective against bacterial isolates and may be added to semen extender to better control bacterial load and semen quality. doi.org/10.54680/fr22610110512.

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching.

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment-protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.

The roles of photorespiration and alternative electron acceptors in the responses of photosynthesis to elevated temperatures in cowpea.

We explored the effects, on photosynthesis in cowpea (Vigna unguiculata) seedlings, of high temperature and light-environmental stresses that often co-occur under field conditions and can have greater impact on photosynthesis than either by itself. We observed contrasting responses in the light and carbon assimilatory reactions, whereby in high temperature, the light reactions were stimulated while CO2 assimilation was substantially reduced. There were two striking observations. Firstly, the primary quinone acceptor (QA ), a measure of the regulatory balance of the light reactions, became more oxidized with increasing temperature, suggesting increased electron sink capacity, despite the reduced CO2 fixation. Secondly, a strong, O2 -dependent inactivation of assimilation capacity, consistent with down-regulation of rubisco under these conditions. The dependence of these effects on CO2 , O2 and light led us to conclude that both photorespiration and an alternative electron acceptor supported increased electron flow, and thus provided photoprotection under these conditions. Further experiments showed that the increased electron flow was maintained by rapid rates of PSII repair, particularly at combined high light and temperature. Overall, the results suggest that photodamage to the light reactions can be avoided under high light and temperatures by increasing electron sink strength, even when assimilation is strongly suppressed.

Multilevel effects of light on ribosome dynamics in chloroplasts program genome-wide and psbA-specific changes in translation.

Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.

Development of a molecular biological assay for the detection of markers related to decreased susceptibility to macrolides and lincomycin in Mycoplasma hyorhinis.

The control of Mycoplasma hyorhinis infection relies mainly on antimicrobial therapy. However, the antibiotic susceptibility testing of the bacteria is usually not performed before applying the treatment, and thus therapeutic failures are not uncommon. In the case of M. hyorhinis, several antibiotic-resistance-related single nucleotide polymorphisms (SNPs) are known but assays for their detection have not been described yet. The aims of the present study were to investigate macrolide- and lincomycin-resistance-related SNPs in Hungarian M. hyorhinis isolates and to develop mismatch amplification mutation assays (MAMA) to detect the identified resistance markers. Minimal inhibitory concentrations (MIC) of different drugs and whole genome sequences of 37 M. hyorhinis isolates were used to find the resistance-related mutations. One MAMA assay was designed to detect the mutation of the 23S rRNA gene at nucleotide position 2058 (Escherichia coli numbering). For further evaluation, the assay was challenged with 17 additional isolates with available MIC data and 15 DNA samples from clinical specimens. The genotypes of the samples were in line with the MIC test results. The developed assay supports the practice of targeted antibiotic usage; hence it may indirectly reduce some bacterial resistance-related public health concerns.

Different Reaction Specificities of F420H2-Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets.

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorum-sensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of l-tyrosine- or l-leucine-derived 4-alkyl-l-proline derivatives (APDs) in their structures. Here, we report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420H2-dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosyntheses of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4-substituted Δ1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, our bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated l-proline-like precursors and likely in the microbial central metabolism.

Chloramphenicol enhances Photosystem II photodamage in intact cells of the cyanobacterium Synechocystis PCC 6803.

The effect of chloramphenicol, an often used protein synthesis inhibitor, in photosynthetic systems was studied on the rate of Photosystem II (PSII) photodamage in the cyanobacterium Synechocystis PCC 6803. Light-induced loss of PSII activity was compared in the presence of chloramphenicol and another protein synthesis inhibitor, lincomycin, by measuring the rate of oxygen evolution in Synechocystis 6803 cells. Our data show that the rate of PSII photodamage was significantly enhanced by chloramphenicol, at the usually applied 200 µg mL-1 concentration, relative to that obtained in the presence of lincomycin. Chloramphenicol-induced enhancement of photodamage has been observed earlier in isolated PSII membrane particles, and has been assigned to the damaging effect of chloramphenicol-mediated superoxide production (Rehman et al. 2016, Front Plant Sci 7:479). This effect points to the involvement of superoxide as damaging agent in the presence of chloramphenicol also in Synechocystis cells. The chloramphenicol-induced enhancement of photodamage was observed not only in wild-type Synechocystis 6803, which contains both Photosystem I (PSI) and PSII, but also in a PSI-less mutant which contains only PSII. Importantly, the rate of PSII photodamage was also enhanced by the absence of PSI when compared to that in the wild-type strain under all conditions studied here, i.e., without addition and in the presence of protein synthesis inhibitors. We conclude that chloramphenicol enhances photodamage mostly by its interaction with PSII, leading probably to superoxide production. The presence of PSI is also an important regulatory factor of PSII photodamage most likely via decreasing excitation pressure on PSII.

Interference with plastome gene expression and Clp protease activity in Arabidopsis triggers a chloroplast unfolded protein response to restore protein homeostasis.

Disruption of protein homeostasis in chloroplasts impairs the correct functioning of essential metabolic pathways, including the methylerythritol 4-phosphate (MEP) pathway for the production of plastidial isoprenoids involved in photosynthesis and growth. We previously found that misfolded and aggregated forms of the first enzyme of the MEP pathway are degraded by the Clp protease with the involvement of Hsp70 and Hsp100/ClpC1 chaperones in Arabidopsis thaliana. By contrast, the combined unfolding and disaggregating actions of Hsp70 and Hsp100/ClpB3 chaperones allow solubilization and hence reactivation of the enzyme. The repair pathway is promoted when the levels of ClpB3 proteins increase upon reduction of Clp protease activity in mutants or wild-type plants treated with the chloroplast protein synthesis inhibitor lincomycin (LIN). Here we show that LIN treatment rapidly increases the levels of aggregated proteins in the chloroplast, unleashing a specific retrograde signaling pathway that up-regulates expression of ClpB3 and other nuclear genes encoding plastidial chaperones. As a consequence, folding capacity is increased to restore protein homeostasis. This sort of chloroplast unfolded protein response (cpUPR) mechanism appears to be mediated by the heat shock transcription factor HsfA2. Expression of HsfA2 and cpUPR-related target genes is independent of GUN1, a central integrator of retrograde signaling pathways. However, double mutants defective in both GUN1 and plastome gene expression (or Clp protease activity) are seedling lethal, confirming that the GUN1 protein is essential for protein homeostasis in chloroplasts.

Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus.

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.