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Wastewater with residual streptomycin sulphate usually contains high content of ammonia-nitrogen. However, the biological removal process of ammonia-nitrogen under streptomycin sulphate circumstance was unclear. In this study, short-term and long-term effects of streptomycin sulphate on biological nitrification systems, including AOB, NOB, SAOR, SNOR and SNPR, were evaluated comprehensively. The results indicated IC50 for AOB and NOB were 7.5 and 6.6 mg/L. SAOR and SNPR could be decreased to 3.43 ± 0.52 mg N/(g MLSS·h) and 0.24 ± 0.03 mg N/(g MLSS·h) while the addition of streptomycin sulphate was 10 mg/L. When streptomycin sulphate addition was stopped, nitrification ability recovered slightly, SAOR and SNPR increased to 9.37 ± 0.36 mg N/(g MLSS·h) and 1.66 ± 0.49 mg N/(g MLSS·h), respectively. The protein of EPS increased gradually during the acclimatization process, and the maximal protein value was 68.24 mg/g MLSS on the 100th day, however, no significant change of polysaccharose was observed during the acclimatization process. High abundance of ARGs and intI1 was detected in effluent and sludge of the biological treatment system. The maximal relative abundance of aadA1 in the sludge appeared on the 140th day, and increased by 0.99 orders of magnitude. Biological diversity decreased significantly during the acclimatization process, relative abundance of nitrosomonas was changed from 9.07% to 38.68% on the 61st day, while relative abundance of nitrobacter was changed from 1.30% to 0.64%. It should be noted that relative abundances of nitrosomonas and nitrobacter were reduced to 16.17% and 0.25% on the 140th day. This study would be helpful for nitrogen removal in wastewater with antibiotic.

Studies have proven that gut microbiota dysbiosis may influence the carcinogenesis and outcomes of multiple cancers. However, it is still unclear whether gut microbiota dysbiosis affect the progression of breast cancer, especially triple-negative breast cancer. In the present study, by using gut microbiota dysbiosis murine model established by treatment of mice with streptomycin, we found Lactobacillus and the metabolite-lactic acid are the pivotal factors for 4T1 tumor progression. In fact, streptomycin-treated mice exhibited slower tumor growth, in parallel with less abundance of Lactobacillus in the gut. Supplementation with Lactobacillus resulted in a rapid tumor growth, following a decrease in the expression of mRNAs for anti-tumor-related factors but an increase in the M2 polarization. The elevated percentages of IFN-γ-producing CD4 + T cells and CD8 + T cells in the tumor microenvironment of streptomycin-treated tumor-bearing mice may be vanished by supplementation of Lactobacillus. It seems likely that lactobacillus-mediated pro-tumor effect is related to the production of lactic acid. A decrease in the levels of lactic acid in the cecal feces and tumor tissues were observed in streptomycin-treated tumor bearing mice. However, supplementation of Lactobacillus can restore streptomycin-reduced concentration of lactic acid in the tumor tissues, suggesting that gut Lactobacillus are the source of lactic acid. Bioinformatics analysis result suggests high concentration of lactic acid in tumor sites may be related to the diminished anti-tumor immunity in the TME. This study reveals a correlation between gut Lactobacillus and tumor progression in a murine 4T1 tumor model, providing experimental evidence for clinical treatment of breast cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Non-typhoidal Salmonella serotypes are well adapted to utilize the inflammation for colonization in the mammalian gut mucosa and cause loss of the integrity of the epithelial barrier in the mammalian intestine. The present study assessed the protective efficacy of fish oil-in-water nanoemulsion, compared to the conventional emulsion, towards the intestinal epithelial barrier against invasive infection of Salmonella enterica serovar Typhimurium strain SL1344 in an in vivo streptomycin-treated mouse model. Non-typhoidal Salmonella enterica serovar Typhimurium strain SL1344 expresses its invasiveness by creating extreme inflammatory assault in the mammalian host lumen via its repertoire of secretory or membrane-bound proteins. Prophylactic treatment of ω-3 polyunsaturated fatty acid-rich fish oil nanoemulsion not only reduced the inflammatory markers by 4-5 fold against the established infection but also retained the gut barrier efficiency as shown by FITC-dextran permeability assay. Though the conventional emulsion also showed similar trends, the efficacy was significantly better with nanoemulsion treatment but neither the nanoemulsion nor conventional emulsion caused any significant change in the microbial colonization of the murine gut mucosa. Mechanistic assessment of the nanoemulsion against inflammation and invasion across the Caco-2 cell monolayer revealed that nanoemulsion treatment protected the expression of Zona occludens-1 along the tight junction, almost by 3-fold as compared to the infected cell monolayer. Such protection was evinced by the trans-epithelial electrical resistance value and the FITC-dextran permeability analysis as well. Fish oil nanoemulsion treatment has also shown significant reduction in pro-inflammatory cytokine expression by the Salmonella strain SL1344 infected Caco-2 cell monolayer. Conventional emulsion also showed distinct protection, but the nanoemulsion offered better protection at the same dosage of fish oil, probably due to its better bioavailability. The results proved that fish oil-loaded nanoemulsion can be efficacious towards maintaining the barrier function and protecting against systemic bacteremia during invasive intestinal infection.

Abstract: Streptomycin is an aminoglycoside antibiotic with a well-known antituberculosis activity; it is commonly used in clinical practice because it is effective and cheap. However, streptomycin has severe ototoxic effects. The delayed and gradual onset may suggest that a metabolic derivative of the antibiotic could be a potential contributor to ototoxicity. As in a rat experimental model this compound was found to be streptidine, we investigated whether this ototoxic metabolite was also present in the blood of streptomycin-treated patients. To this end, we implemented and optimized a direct reverse-phase HPLC technique to identify and estimate streptomycin and streptidine in serum of streptomycin-treated patients. All criteria for validation of the method were implemented in standard curves in serum of healthy non-treated volunteers by addition of increasing concentration of both compounds and their determination in a trichloroacetic acid deproteinized extract. We found that recovery of streptomycin or streptidine was ≥91.5%. Linearity was r 2 ≥0.99. The intraday and interday precisions were ≤9.7 and ≤10.6%, respectively. The relative intraday and interday error ranged from −9.0 to 8.3% for both compounds in human serum. Studies in patients included five male individuals treated from 35 to 90 days with 1g/day of streptomycin, presenting inner ear malfunction from mild to severe, in whose serum streptidine was always present, and could be successfully separated from streptomycin. Therefore, the validated method used can be a valuable tool to measure and follow these compounds in serum of streptomycin-treated patients. [Copyright &y& Elsevier]

The MtrCDE system confers multidrug resistance to Neisseria gonorrhoeae , the causative agent of gonorrhea. Using free and directed molecular dynamics (MD) simulations, we analyzed the interactions between MtrD and azithromycin, a transport substrate of MtrD, and a last-resort clinical treatment for multidrug-resistant gonorrhea. We then simulated the interactions between MtrD and streptomycin, an apparent nonsubstrate of MtrD. Using known conformations of MtrD homologues, we simulated a potential dynamic transport cycle of MtrD using targeted MD techniques (TMD), and we noted that forces were not applied to ligands of interest. In these TMD simulations, we observed the transport of azithromycin and the rejection of streptomycin. In an unbiased, long-time scale simulation of AZY-bound MtrD, we observed the spontaneous diffusion of azithromycin through the periplasmic cleft. Our simulations show how the peristaltic motions of the periplasmic cleft facilitate the transport of substrates by MtrD. Our data also suggest that multiple transport pathways for macrolides may exist within the periplasmic cleft of MtrD.

Certain point mutations within gene for ribosomal protein S12, rpsL, are known to dramatically change physiological traits of bacteria, most prominently antibiotic resistance and production of various metabolites. The rpsL mutants are usually searched among spontaneous mutants resistant to aminoglycoside antibiotics, such as streptomycin or paromomycin. The shortcomings of traditional selection are as follows: random rpsL mutants may carry undesired genome alterations; many rpsL mutations cannot be isolated because they are either not associated with increased antibiotic resistance or non-viable in the absence of intact rpsL WT gene. Introduction of mutant rpsL alleles in the rpsL WT background can be used to circumvent these obstacles. Here we take the latter approach and report the generation and properties of a set of stable rpsL merodiploids for Streptomyces albus J1074. We identified several rpsL alleles that enhance endogenous and heterologous antibiotic production by this strain and show that rpsL WT rpsL K88E merodiploid displays increased streptomycin resistance. We further tested several promising rpsL alleles in two more strains, Streptomyces cyanogenus S136 and Streptomyces ghanaensis ATCC14672. In S136, plasmid-borne rpsL K88E+P91S and rpsL K88R led to elevated landomycin production; no changes were detected for ATCC14672 merodiploids. Our data outline the prospects for and limitations to rpsL merodiploids as a tool for rapid enhancement of secondary metabolism in Streptomyces.

Here, we investigate the unfolding behavior of a streptomycin-binding ribonucleic acid (RNA) aptamer under application of force in shear geometry. Using Langevin out-of-equilibrium simulations to emulate the single-molecule force spectroscopy (SMFS) experiment, we were able to understand the hierarchical unfolding process that occurs in the RNA molecule under application of stretching force and the influence of streptomycin modifying this unfolding. Subsequently, the application of the Jarzynski equality to the force profiles obtained in the pulling simulations shows that the free energies for individual systems and the difference of unfolding free energy upon streptomycin binding to the RNA free aptamer are in fair agreement with the experimental values, obtained through SMFS by Nick et al. [J. Phys. Chem. B 120, 6479 (2016)].

Despite advances, tuberculosis remains a significant infectious disease, whose mortality presents alarming numbers. Although it can be cured, the number of cases of antimicrobial resistant strains is increasing, requiring the use of less efficient second-line drugs. Capreomycin and streptomycin are part of this group, being antibiotics whose mechanism of action is the inhibition of protein synthesis when interacting with the tuberculosis bacterial ribosome. Their binding mechanisms are distinct: capreomycin is able to bind to both ribosomal (30S and 50S) subunits, whereas streptomycin binds only to the smaller one (30S). In this context, the biochemical characterization of these binding sites for a proper understanding of their complex interactions is of crucial importance to increase their efficacy. Through crystallographic data and computer simulations, in this work we calculated the interaction binding energies of capreomycin and streptomycin in complex with the tuberculosis bacterial ribosome subunits, by using density functional theory (DFT) within the molecular fractionation with conjugated caps (MFCC) approach. For capreomycin in the 30S (50S) subunit, we investigated the binding energies of 44 (30) residues presented within a pocket radius of 14 Å (30 Å). Regarding streptomycin, 60 nucleotide (25 amino acid) residues distributed up to 12.5 Å (15 Å) away from the drug in the 30S subunit (S12 protein) were taken into account. We also identify the contributions of hydrogen bonds and hydrophobic interactions in the drug-receptor complex, and the regions of the drugs that most contributed to the anchorages of them in their binding sites, as well as identify residues that are most associated with mutations.

An electrochemical method is described for the determination of streptomycin (STR). It is making use of a gold electrode coated with a thin mesoporous silica film (MSF). In addition, silver nanoparticles were coated on the MSF to increase the surface area, to bind a large amount of aptamer (Apt), and to improve the electrical conductivity. In the presence of STR, it will bind to the Apt and hinder the diffusion of the redox probe hexacyanoferrate through the nanochannels of the mesoporous film. The aptasensor, best operated at a working potential of 0.22 V (vs. Ag/AgCl) has a linear response in the 1 fg.mL -1 to 6.2 ng.mL -1 STR concentration range. The detection limit is 0.33 fg.mL -1 . The assay was successfully validated by analyzing spiked samples of milk and blood serum. Graphical abstract Voltammetric assay of streptomycin (STR) by using a Fe(CN) 6 3-/4- probe. The aptamer was immobilized on a gold electrode modified with a mesoporous silica thin film (MSF) that was functionalized with (3-aminopropyl) triethoxysilane (APTES) and silver nanoparticles (AgNP). Incubation with STR leads to a decrease of the current.

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O- adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin., (© 2018 Stern et al.)

This paper describes the extracellular synthesis of silver nanoparticles from waste part of lychee fruit (peel) and their conjugation with selected antibiotics (amoxicillin, cefixim, and streptomycin). FTIR studies revealed the reduction of metallic silver and stabilization of silver nanoparticles and their conjugates due to the presence of CO (carboxyl), OH (hydroxyl) and CH (alkanes) groups. The size of conjugated nanoparticles varied ranging from 3 to 10 nm as shown by XRD. TEM image revealed the spherical shape of biosynthesized silver nanoparticles. Conjugates of amoxicillin and cefixim showed highest antibacterial activity (147.43 and 107.95%, respectively) against Gram-negative bacteria i.e. Alcaligenes faecalis in comparison with their control counterparts. The highest reduction in MIC was noted against Gram-positive strains i.e. Enterococcus faecium (75%) and Microbacterium oxydans (75%) for amoxicillin conjugates. Anova two factor followed by two-tailed t test showed non-significant results both in case of cell leakage and protein estimation between nanoparticles and conjugates of amoxicillin, cefixime and streptomycin. In case of MDA release, non-significant difference among the test samples against the selected strains. Our study found green-synthesized silver nanoparticles as effective antibacterial bullet against both Gram positive and Gram negative bacteria, but they showed a more promising effect on conjugation with selected antibiotics against Gram negative type.

Pulmonary intracellular infections, such as tuberculosis, anthrax, and tularemia, have remained a significant challenge to conventional antibiotic therapy. Ineffective antibiotic treatment of these infections can lead not only to undesired side effects, but also to the emergence of antibiotic resistance. Aminoglycosides (e.g., streptomycin) have long been part of the therapeutic regiment for many pulmonary intracellular infections. Their bioavailability for intracellular bacterial pools, however, is limited by poor membrane permeability and rapid elimination. To address this challenge, polymer-augmented liposomes (PALs) were developed to provide improved cytosolic delivery of streptomycin to alveolar macrophages, an important host cell for intracellular pathogens. A multifunctional diblock copolymer was engineered to functionalize PALs with carbohydrate-mediated targeting, pH-responsive drug release, and endosomal release activity with a single functional polymer that replaces the pegylated lipid component to simplify the liposome formulation. The pH-sensing functionality enabled PALs to provide enhanced release of streptomycin under endosomal pH conditions (70% release in 6 hours) with limited release at physiological pH 7.4 (16%). The membrane-destabilizing activity connected to endosomal release was characterized in a hemolysis assay and PALs displayed a sharp pH profile across the endosomal pH development target range. The direct connection of this membrane-destabilizing pH profile to model drug release was demonstrated in an established pyranine/p-xylene bispyridinium dibromide (DPX) fluorescence dequenching assay. PALs displayed similar sharp pH-responsive release, whereas PEGylated control liposomes did not, and similar profiles were then shown for streptomycin release. The mannose-targeting capability of the PALs was also demonstrated with 2.5 times higher internalization compared to non-targeted PEGylated liposomes. Finally, the streptomycin-loaded PALs were shown to have a significantly improved intracellular antibacterial activity in a Francisella-macrophage co-culture model, compared with free streptomycin or streptomycin delivered by control PEGylated liposomes (13× and 16×, respectively). This study suggests the potential of PALs as a useful platform to deliver antibiotics for the treatment of intracellular macrophage infections.

Covering: 2000 to 2018 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.

Oral bioavailability of a drug molecule requires its effective delivery to the target site. In general, majority of synthetically developed molecular entities have high hydrophobic nature as well as low bioavailability, therefore the need for suitable delivery vehicles arises. Self-assembled structures such as micelles, niosomes, and liposomes have been used as effective delivery vehicles and studied extensively. However, the information available in literature is mostly qualitative in nature. We have quantitatively investigated the partitioning of antibiotic drug streptomycin into cationic, nonionic, and a mixture of cationic and nonionic surfactant micelles and its interaction with the transport protein serum albumin upon subsequent delivery. A combination of calorimetry and spectroscopy has been used to obtain the thermodynamic signatures associated with partitioning and interaction with the protein and the resulting conformational changes in the latter. The results have been correlated with other class of drugs of different nature to understand the role of molecular features in the partitioning process. These studies are oriented toward understanding the physical chemistry of partitioning of a variety of drug molecules into suitable delivery vehicles and hence establishing structure-property-energetics relationships. Such studies provide general guidelines toward a broader goal of rational drug design.

The secondary metabolite acarbose is used worldwide in the clinical treatment of diabetes mellitus type 2 patients. Acarbose is a - glucosidase inhibitor and supports patients to control their blood glucose as well as their serum insulin levels. The secondary metabolite is produced by strains of the class Actinobacteria, in particular from Actinoplanes sp. SE50/110, which is a progenitor of today`s production strains. Moreover, secondary metabolite clusters could also be identified in Streptomyces coelicoflavus ZG0656 as well as Streptomyces glaucescens GLA.O. In this study, the genome S. glaucescens GLA.O with focus on the acarbose biosynthesis cluster (gac-cluster) was analyzed. First, the tetracenomycin C and the 5`-hydroxy streptomycin gene clusters could be described completely. Then the gac gene region in S. glaucescens GLA.O is compared to the other known biosynthesis gene cluster. In comparison to Actinoplanes sp. SE50/110 the gac-cluster showed structural variances, like the missing homolog of the glycosyltransferase AcbD in the whole genome of S. glaucescens GLA.O. Due to the lack of the glycosyltransferase, it was of particular interest whether additional acarviose metabolites other than acarbose could be formed. For detection of acarviose metabolites biosynthesis the supernatant of S. glaucescens GLA.O grown in starch supplemented complex media was harvested at 72 and 96 hours. Although a homolog of the known glycosyltransferase is absent, the LC-MS-supported analysis revealed that a spectrum of acarviose metabolites was formed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (But R ) and of the str mutation (Sm R ; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the But R Sm R mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The emergence and spread of antibiotic resistance among Acinetobacter spp. have been investigated extensively. Most studies focused on the multiple antibiotic resistance genes located on plasmids or genomic resistance islands. On the other hand, the mechanisms controlling intrinsic resistance are still not well understood. In this study, we identified the novel subclass of aminoglycoside nucleotidyltransferase ANT(3")-II in Acinetobacter spp., which comprised numerous variants distributed among three main clades. All members of this subclass can inactivate streptomycin and spectinomycin. The three ant(3")-II genes, encoding for the three ANT(3")-II clades, are widely distributed in the genus Acinetobacter and always located in the same conserved genomic region. According to their prevalence, these genes are intrinsic in Acinetobacter baumannii, Acinetobacter pittii, and Acinetobacter gyllenbergii. We also demonstrated that the ant(3")-II genes are located in a homologous recombination hotspot and were recurrently transferred among Acinetobacter species. In conclusion, our findings demonstrated a novel mechanism of natural resistance in Acinetobacter spp., identified a novel subclass of aminoglycoside nucleotidyltransferase and provided new insight into the evolutionary history of intrinsic resistance genes.

Studies have shown that external stress induces biofilm formation, but the underlying details are not clearly understood. This study investigates the changes in cell surface properties leading to increase in biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa in the presence of streptomycin. Bacterial attachment in the presence and absence of streptomycin was quantified by fluorescence spectroscopy. In addition, cell surface charge and contact angle were measured and the free energy barrier for attachment was modeled using extended Derjaguin-Landau-Verwey-Overbeek (xDLVO) theory. Peptides from bacterial cell surface were shaved by protease treatment and identified with ultra-performance liquid chromatography (UPLC)-QTOF and a homology search program SPIDER. Biofilm formation increased significantly in the presence of streptomycin (10 mg/L) in the culture. Bacterial cell surface charge reduced, and hydrophobicity increased leading to a net decrease in the free energy barrier for attachment. Extracellular matrix-binding protein was positively regulated in S. aureus under stress, indicating stronger interaction between bacterial cells and solid surface. In addition, several other proteins including biofilm regulatory proteins, multidrug efflux pumps, transporters, signaling proteins, and virulence factors were differentially expressed on bacterial cell surface, which is indicative of a strong stress response by bacteria to streptomycin treatment.

Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms.

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.

Unlabelled: Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as "colonization resistance." However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment., Importance: Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers "colonization resistance" through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

Antibiotic resistance concerns have been compounded by a report that soil bacteria can catabolise antibiotics, i.e. break down and use them as a sole carbon source. To date this has not been verified or reproduced, therefore in this study soil bacteria were screened to verify and reproduce this hypothesis. Survival in high concentrations of antibiotics was initially observed; however, on further analysis these bacteria either did not degrade the antibiotics or they used an intrinsic resistance mechanism (β-lactamases) to degrade the β-lactams, as demonstrated by high-performance liquid chromatography. These results did not verify or reproduce the hypothesis that bacteria subsist on antibiotics or catabolise antibiotics as previously reported. This study identified that bacteria with a catabolising phenotype did not degrade streptomycin or trimethoprim and therefore could not utilise the antibiotics as a nutrient source. Therefore, we conclude that soil bacteria do not catabolise antibiotics., (Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

In the underwater environment, sound propagates both as a pressure wave and as particle displacement, with particle displacement dominating close to the source (the nearfield). At the receptor level, both the fish ear and the neuromast hair cells act as displacement detectors and both are potentially stimulated by the particle motion component of sound sources, especially in the nearfield. A now common way to test 'hearing' in fish involves auditory evoked potentials (AEPs), with recordings made from electrodes implanted near the auditory brainstem. These AEP recordings are typically conducted in enclosed acoustic environments with the fish well within the nearfield, especially for lower frequencies. We tested the contribution of neuromast hair cells to AEP by first testing intact goldfish (Carassius auratus), then ablating their neuromasts with streptomycin sulphate--disabling superficial and canal neuromasts--and retesting the same goldfish. We performed a similar experiment where only the superficial neuromasts were physically ablated. At 100 and 200 Hz, there was a 10-15 dB increase in threshold after streptomycin treatment but no significant difference at higher frequencies. There was no difference in threshold in control fish or in fish that only had superficial neuromasts removed, indicating that the differential responses were driven by canal neuromasts. Taken together, these results indicate that AEP results at lower frequencies should be interpreted as multimodal responses, rather than as 'hearing'. The results also suggest that in natural situations both the ear and lateral line likely play an integrative role in detecting and localising many types of 'acoustic' stimuli.

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9-13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Mycobacterium tuberculosis 18b, a streptomycin (STR)-dependent mutant that enters a viable but nonreplicating state in the absence of STR, has been developed as a simple model for drug testing against dormant bacilli. Here, we further evaluated the STR-starved 18b (SS18b) model both in vitro and in vivo by comparing the behavior of 22 approved and experimental tuberculosis drugs. Using the resazurin reduction microplate assay (REMA), rifampin (RIF), rifapentine (RPT), TMC207, clofazimine (CFM), and linezolid (LIN) were found to be active against SS18b in vitro, and their bactericidal activity was confirmed by determining the number of CFU. A latent 18b infection was established in mice, and some of the above-mentioned drugs were used for treatment, either alone or in combination with RIF. RIF, RPT, TMC207, CFM, and pyrazinamide (PZA) were all active in vivo, while cell wall inhibitors were not. A comparative kinetic study of rifamycin efficacy was then undertaken, and the results indicated that RPT clears latent 18b infection in mice faster than RIF. Intrigued by the opposing responses of live and dormant 18b cells to cell wall inhibitors, we conducted a systematic analysis of 14 such inhibitors using REMA. This uncovered an SS18b signature (CWPRED) that accurately predicted the activities of cell wall inhibitors and performed well in a blind study. CWPRED will be useful for establishing the mode of action of compounds with unknown targets, while the SS18b system should facilitate the discovery of drugs for treating latent tuberculosis.

Evidence for ageing in symmetrically dividing bacteria such as Escherichia coli has historically been conflicting. Early work found weak or no evidence. More recent studies found convincing evidence, but negative results are still encountered. Because bacterial ageing is believed to result from non-genetic (e.g. oxidative) damage, we tested the possibility that the negative outcomes resulted from the lack of an extrinsic damage agent. We found that streptomycin, which produces mistranslated proteins that are more vulnerable to oxidation, was able to induce both damage and ageing in bacterial populations. A dosage effect relating the level of damage to the concentration of streptomycin was observed. Our results explain the previous inconsistencies, because all studies that failed to find evidence for bacterial ageing did not use a damage agent. However, all studies that succeeded in finding evidence utilized fluorescent proteins as a visual marker. We suggest that ageing in those studies was induced by the harmful effects of an extrinsic factor, such as the proteins themselves or the excitation light. Thus, all of the earlier studies can be reconciled and bacterial ageing is a real phenomenon. However, the study and observation of bacterial ageing require the addition of an extrinsic damage agent.

Bacillus subtilis induces expression of the gene ytnP in the presence of the antimicrobial streptomycin, produced by the Gram-positive bacterium Streptomyces griseus. ytnP encodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium in S. griseus.

The mechanism of the competitive interaction of streptomycin and Evans blue (EB) to bovine serum albumin (BSA) has been studied by using both fluorimetry and spectrophtometry. Effects of pH, streptomycin and concentration of EB on the competitive interaction of streptomycin and EB were examined. A static fluorescence quenching process was confirmed in the light of Stern-Volmer plot. The test result showed that there were strong and weak binding sites on BSA molecule and the binding constant of EB-BSA complex and the number of binding site n were obtained. These facts revealed that the competitive interaction was occurred between EB and streptomycin, which can possibly provide useful message in investigation of the interaction of antibiotic with BSA., (Copyright © 2011 Elsevier B.V. All rights reserved.)

AdpA is a global transcriptional regulator that is induced by the microbial hormone A-factor and activates many genes required for morphological differentiation and secondary metabolism in Streptomyces griseus. We confirmed that the regulatory tRNA gene bldA was required for translation of TTA-containing adpA. We also demonstrated that AdpA bound two sites upstream of the bldA promoter and activated transcription of bldA. Thus, we revealed a unique positive feedback loop between AdpA and BldA in S. griseus. Forced expression of bldA in an A-factor-deficient mutant resulted in the partial restoration of aerial mycelium formation and streptomycin production, suggesting that the positive feedback loop could prevent premature transcriptional activation of the AdpA-target genes in the wild-type strain. We revealed that the morphological defect of the bldA mutant could be attributed mainly to the TTA codons of only two genes: adpA and amfR. amfR encodes a transcriptional activator essential for aerial mycelium formation and is a member of the AdpA regulon. Thus, amfR is regulated by a feedforward mechanism involving AdpA and BldA. We concluded that the central regulatory unit composed of AdpA and BldA plays important roles in the initiation of morphological differentiation and secondary metabolism triggered by A-factor., (© 2011 Blackwell Publishing Ltd.)

Origins of antibiotic drug discovery are frequently traced to 1929 when Alexander Fleming recognized the antibacterial activity of a substance secreted by Penicillium notatum on a contaminated culture plate. However, the subsequent development of penicillin as a therapeutic agent was not realized until the early 1940s, after a consortium of academic and pharmaceutical scientists from England and the United States developed sufficiently advanced fermentation technology to produce high-purity penicillin in large enough quantities for medical supplies. It was at this time that the antibiotic era was truly successfully launched. During the following decade, unprecedented antibiotic research and development emerged in academic laboratories and the pharmaceutical industry, resulting in identification of most of the antibiotic classes currently used therapeutically. This short historical commentary describes some of these early events, beginning with a conference held at the New York Academy of Sciences in 1946, the first conference to focus entirely on the latest science related to the identification and characterization of antibacterial substances produced by microorganisms.

Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD-fused Strep-Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of Strep-tag II-linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin-introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for Strep-tag II-fused cargos.

For drugs that target the central nervous system (CNS) the penetration of the blood-brain barrier (BBB) is a necessity, whereas for other drugs it is an unwanted side-effect. Infections by Staphylococcus aureus (SA) of the CNS causes brain abscesses and inflammation of the tissue enclosing the brain and spinal cord. In this study four sulfonamide agents are compared to streptomycin for growth inhibition of SA bacteria in vitro. All sulfonamide agents were synthesized using microwave irradiation techniques. All drugs tested show substantial growth inhibition of MRSA and MSSA types of Staphylococcus aureus. Important pharmacological properties of streptomycin and sulfonamide drugs are determined to evaluate the efficacy for CNS targeting in clinical treatment of MSSA and and MRSA infections. Although streptomycin demonstrated considerable inhibition of MSSA and MRSA bacteria, the Log P value of -5.28 and polar surface area (PSA) of 331.4 Angstroms(2) effectively inhibits its penetration into the CNS. The Log BB for streptomycin at -4.756 also indicates a very unlikely candidate to treat SA infections of the CNS. All sulfonamide agents showed significant growth inhibition of MRSA and MSSA while having useful values of Log P and PSA for penetrating the BBB. In addition the Log BB values for each sulfonamide agent indicates these agents will enter the CNS to halt SA infections. The Log BB values for sulfonamide drugs 2, A, B, and C are -1.658, -0.109, -0.109, and -0.243, respectively. Values for Log P were 2.549, 3.27, 3.47, and 2.63, respectively; with PSA values at 137.8 A(2), 40.98 A(2), 40.98 A(2), and 49.36 A(2), respectively. The small size of the sulfonamide agents and other physicochemical properties strongly support the contention they can be effectively applied to treat MRSA and MSSA infections of the brain and spinal cord.

The RsmG methyltransferase is responsible for N(7) methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 A resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.

Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays.

Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

Although guinea pigs are considered one of the best animal models of tuberculosis, little data exist describing latent or dormant tuberculosis infection in these animals. Here we address this issue using a streptomycin auxotrophic mutant of Mycobacterium tuberculosis. This mutant grows unimpaired in the presence of streptomycin but in its absence shifts to latency/dormancy (lack growth and over-expression of alpha-crystallin). To establish infection animals are inoculated with the mutant followed by daily administration of streptomycin (three weeks), which allows initial microbial multiplication in the animal's tissues. Withdrawal of streptomycin establishes latency/dormancy and few viable organisms are recovered from the animals' lungs and spleen six months later. During the infectious process guinea pigs steadily gained weight and presented no clinical signs (scuff fur and lethargy) of disease. Histopathology of organs mimicked tuberculous lesions in humans and PBMC from infected animals strongly responded to stimulation with PPD. Finally, tuberculin skin test (a hallmark of latent infection diagnosis) performed in infected animals was strongly positive (>or=15 mm induration). These results point to an interesting and reliable model of latent/dormant tuberculosis infection in guinea pigs.

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

MexXY is an active efflux system that contributes to intrinsic resistance to aminoglycosides in Pseudomonas aeruginosa. MexXY can function in combination with OprM in aminoglycoside efflux but may also functionally associate with another as yet unidentified outer membrane channel. The possible role of OpmG as a third component of MexXY in aminoglycoside efflux was investigated by construction of unmarked opmG mutants. Loss of OpmG did not have any impact on minimum inhibitory concentrations for aminoglycosides regardless of the presence of oprM, indicating that MexXY does not interact with OpmG in aminoglycoside efflux. In a clinical isolate PAJ010, (mexXY) enhanced streptomycin susceptibility but neither oprM nor opmG could, suggesting that MexXY functionally associates with an unidentified outer membrane protein for aminoglycoside efflux. Expression of an opmG-lacZ transcriptional fusion revealed that OpmG expression was neither constitutive nor inducible by gentamicin. Growth rates of wildtype P. aeruginosa and opmG mutant derivatives were not different, indicating that expression of opmG is not essential for P. aeruginosa growth.

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.

In Streptomyces coelicolor, AtrA is an activator of transcription of the actinorhodin cluster-situated regulator gene actII-ORF4. In previous work, we showed that S. coelicolor AtrA binds in vitro to the promoter of S. griseus strR, the streptomycin cluster-situated regulator. We show here that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA in S. griseus causes a DNA binding-dependent reduction in streptomycin production and in the mRNA levels of strR and genes of streptomycin biosynthesis. However, there is no effect on the level of the mRNA of adpA, which is the only transcription factor that has so far been characterised for strR. The adpA gene is directly regulated by ArpA, the receptor protein for the gamma-butyrolactone signalling molecule A-factor. Therefore, to our knowledge, our results provide the first in vivo evidence that A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. The potential biotechnological application of our findings is discussed.

Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1-2 h.

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily by enzymic modification of these drugs. One important mechanism of streptomycin modification is through ATP-dependent O-adenylation, catalyzed by streptomycin adenylyltransferase. Initial velocity patterns deduced from steady state kinetics indicate a sequential mechanism. Dead-end inhibition by tobramycin and neomycin is non-competitive versus streptomycin and uncompetitive versus ATP, indicative of ordered substrate binding where ATP binds first and then streptomycin. These results surmise that streptomycin adenylyltransferase follows an ordered, sequential kinetic mechanism in which one substrate (ATP) binds prior to the antibiotic and pyrophosphate is released prior to formation of AMP-streptomycin.

Streptomycin is the treatment of choice in developing countries for patients suffering from tuberculosis or other infectious diseases. However, it produces incapacitating vestibular symptoms whose onset is delayed and gradual. This observation led to the notion that a streptomycin metabolic derivative and not the antibiotic itself is the damaging agent for the inner ear. To study further the existence of this ototoxic metabolite, chronic treatment with streptomycin or its putative derivative streptidine was carried out in young male Long Evans rats. The presence of streptomycin or streptidine in the blood of animals of either experimental group was assessed by high performance liquid chromatography and analysis of swimming behavior was used to evaluate vestibular damage. Features of the sensory epithelium and quantification of hair cells were attained in sections of the utricular organ of all groups by light microscopy. After 25, 35 and 45 days of treatment with streptomycin, a metabolite with the same chromatographic properties as the streptidine standard run in parallel was identified in the blood of rats. Concentrations of the metabolite were 2.26 microg/ml on the 25th day and around 8.0 microg/ml in both the 35th and the 45th day of treatment, while streptomycin was below its detection level at either period. In streptidine-treated rats, the concentration of this compound was 1.0, 1.84 and 4.94 microg/ml on the 25th, 35th and 45th treatment days, respectively. Treatment with either streptomycin or streptidine resulted in similar abnormal swimming patterns and histological alterations of the utricular epithelium. Loss of hair cells was roughly equivalent even though streptidine was administered in a dose 90% lower than streptomycin. The gradual appearance of streptidine as a metabolic derivative of the antibiotic in the blood of rats or the administration of this compound alone, causing similar functional and structural vestibular deterioration seen in streptomycin-treated animals, supports the notion that streptidine is a potential contributor to ototoxicity after prolonged antibiotic administration.

Multidrug resistance-associated protein 2 (MRP2) is associated with active drug efflux and may influence oral bioavailability of common classes of drugs. MRP2 expression demonstrates plasticity. Caco-2 cells, a routine in vitro model for predicting oral bioavailability, are often cultured in media containing antibiotics. We have investigated whether exposure of Caco-2 cells to two common antibiotic regimes alters MRP2 functional expression. Caco-2 cells were grown in the presence or absence of either gentamicin or penicillin-streptomycin for up to 9 weeks. MRP2 functional activity was assessed by calcein efflux across the apical membrane. MRP2 protein expression was determined by immunoblots. Neither antibiotic regime resulted in consistent changes in calcein efflux across the apical membrane (reflecting MRP2 activity) or basolateral membrane (reflecting MRP3 and possibly MRP6 activity) of Caco-2 cells. MRP2 protein expression also showed no change in response to antibiotic exposure. Routine exposure of Caco-2 cells to penicillin-streptomycin or gentamicin does not affect apical MRP2 functional activity in intestinal enterocytic Caco-2 cells. Extrapolating these results to the situation in vivo suggests that the oral bioavailability of MRP substrates is not predicted to be influenced by recent courses of antibiotics.

In the model of the A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) regulatory cascade in Streptomyces griseus, A-factor binds ArpA, the A-factor receptor protein, that has bound to the adpA promoter and dissociates it from the DNA, thus inducing the transcription of adpA. AdpA switches on the transcription of a number of genes required for secondary metabolism and morphological differentiation, forming an AdpA regulon. Consistent with this model, arpA null mutants produced streptomycin and a yellow pigment in larger amounts and formed aerial hyphae from an earlier growth stage than the wild-type strain. On the other hand, mutant MK2, expressing a mutant ArpA (Trp119Ala), neither produced secondary metabolites nor formed aerial hyphae, because this A-factor-insensitive mutant ArpA always bound to and repressed the adpA promoter due to the amino acid replacement of Trp-119 with Ala. Introduction of adpA under the control of a foreign promoter into mutant MK2 restored all of the phenotypes that we could observe, which suggests that the only significant target of ArpA is adpA. In contrast to other gamma-butyrolactone regulatory systems, disruption of arpA had no effect on A-factor production, indicating that ArpA does not regulate A-factor biosynthesis. Instead, A-factor production was found to be repressed by AdpA in a two-step regulatory feedback loop.