Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics.

Actinobacteria, which are one of the largest bacterial phyla and comprise between 13 and 30% of the soil microbiota, are the main source of antibiotic classes in clinical use1. During screens for antimicrobials, as many as 50% of actinomycete strains are discarded because they produce a known antibiotic (Supplementary Fig. 1) (ref. 2). Despite each strain likely having the capacity to produce many compounds, strains are abandoned because the already characterized antibiotic could interfere with screening for, or purification of, newly discovered compounds3. We applied CRISPR-Cas9 genome engineering to knockout genes encoding two of the most frequently rediscovered antibiotics, streptothricin or streptomycin, in 11 actinomycete strains. We report that this simple approach led to production of different antibiotics that were otherwise masked. We were able to rapidly discover rare and previously unknown variants of antibiotics including thiolactomycin, amicetin, phenanthroviridin and 5-chloro-3-formylindole. This strategy could be applied to existing strain collections to realize their biosynthetic potential.

Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance.

Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.

Catalytic promiscuity of glycopeptide N-methyltransferases enables bio-orthogonal labelling of biosynthetic intermediates.

We show that two α-N-methyltransferases involved in the biosynthesis of glycopeptide antibiotics (GPAs) already recognise partly crosslinked precursor peptides of teicoplanin aglycone indicating that in vivo N-methylation can occur as an early tailoring step during GPA biosynthesis. This relaxed substrate specificity is accompanied by a remarkable promiscuity regarding the co-substrate enabling modulation of biological activity and the introduction of reactive handles which could be further modified using bio-orthogonal chemistry.

How To Make a Glycopeptide: A Synthetic Biology Approach To Expand Antibiotic Chemical Diversity.

Modification of natural product backbones is a proven strategy for the development of clinically useful antibiotics. Such modifications have traditionally been achieved through medicinal chemistry strategies or via in vitro enzymatic activities. In an orthogonal approach, engineering of biosynthetic pathways using synthetic biology techniques can generate chemical diversity. Here we report the use of a minimal teicoplanin class glycopeptide antibiotic (GPA) scaffold expressed in a production-optimized Streptomyces coelicolor strain to expand GPA chemical diversity. Thirteen scaffold-modifying enzymes from 7 GPA biosynthetic gene clusters in different combinations were introduced into S. coelicolor, enabling us to explore the criteria for in-cell GPA modification. These include identifying specific isozymes that tolerate the unnatural GPA scaffold and modifications that prevent or allow further elaboration by other enzymes. Overall, 15 molecules were detected, 9 of which have not been reported previously. Some of these compounds showed activity against GPA-resistant bacteria. This system allows us to observe the complex interplay between substrates and both non-native and native tailoring enzymes in a cell-based system and establishes rules for GPA synthetic biology and subsequent expansion of GPA chemical diversity.

Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA-Specified Leu-tRNA(UUA) Molecule.

Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA-encoded Leu-tRNA(UUA) molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by (19) F NMR spectroscopy, HPLC-ESI-MS, and HPLC-continuum source molecular absorption spectroscopy for fluorine-specific detection. The molecule was purified from a large-scale culture and definitively characterized by NMR spectroscopy and high-resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5'-O-sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60-year-old mystery.

Assessing youth offenders in a non-Western context: The predictive validity of the YLS/CMI ratings.

Empirical support for the usage of the Youth Level of Service measures has been reported in studies conducted in the North America, United Kingdom, and Australia. Recent meta-analytic studies on the Youth Level of Service/Case Management Inventory (YLS/CMI) have revealed that the measure has modest to moderate predictive validity for general recidivism, but there are very few studies on the predictive validity of the YLS/CMI ratings for recidivism in non-Western contexts. This study examined the predictive validity of the YLS/CMI 2.0 ratings for general recidivism in a sample of 3,264 youth offenders within a Singaporean context (Mfollow-up = 1,764.5 days; SDfollow-up = 521.5). Results showed that the YLS/CMI 2.0 overall risk ratings and total scores significantly predicted general recidivism for both male and female youth offenders. Overall, the results suggest that the YLS/CMI 2.0 is suited for assessing youth offenders in terms of their risk for general recidivism within a non-Western context.

Harnessing the synthetic capabilities of glycopeptide antibiotic tailoring enzymes: characterization of the UK-68,597 biosynthetic cluster.

In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes.

Glycopeptide antibiotic biosynthesis.

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

Implementation of the Risk-Need-Responsivity Framework across the Juvenile Justice Agencies in Singapore.

The Risk-Need-Responsivity (RNR) framework is regarded as the forefront of offender rehabilitation in guiding youth offender risk assessment and interventions. This article discusses the juvenile justice system in Singapore and the local research that has been conducted in relation to the RNR framework and the associated Youth Level of Service (YLS) measures. It describes a journey that saw the implementation of the RNR framework across the juvenile justice agencies and highlights the challenges that were faced during the implementation process on the ground. Finally, the article concludes by providing future directions for the implementation of the RNR framework in Singapore.

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

Complex integrons containing qnrB4-ampC (bla(DHA-1)) in plasmids of multidrug-resistant Citrobacter freundii from wastewater.

Microbial populations in wastewater treatment plants (WWTPs) are increasingly being recognized as environmental reservoirs of antibiotic resistance genes. PCR amplicons for plasmid-mediated quinolone resistance determinants qnrA, qnrB, and qnrS were recorded in samples from a WWTP in Vancouver, British Columbia. Six strains of ciprofloxacin-resistant Citrobacter freundii were isolated and found to carry mutations in gyrA and parC, as well as multiple plasmid-borne resistance genes, collectively including qnrB; aac(6')-Ib-cr; ß-lactamase-encoding genes from molecular classes A (blaTEM-1), C (ampC), D (blaOXA-1, blaOXA-10); and genes for resistance to 5 other types of antibiotics. In 3 strains, large (>60 kb) plasmids carried qnrB4 and ampC as part of a complex integron in a 14 kb arrangement that has been reported worldwide but, until recently, only among pathogenic strains of Klebsiella. Analysis of single-nucleotide polymorphisms in the qnrB4-ampC regions infers 2 introductions into the WWTP environment. These results suggest recent passage of plasmid-borne fluoroquinolone and ß-lactam resistance genes from pathogens to bacteria that may be indigenous inhabitants of WWTPs, thus contributing to an environmental pool of antibiotic resistance.

Separate mechanisms are involved in rifampicin upmodulated and downmodulated gene expression in Salmonella Typhimurium.

Sub-MIC antibiotics differentially modulate transcription of subsets of genes by unknown mechanisms. Paradoxically, the RNA polymerase inhibitor rifampicin is able to both upmodulate as well as downmodulate transcription when present at sub-MIC levels. In this study, we analyzed DNA sequences required for transcription modulation. For three downmodulated promoters, the necessary sequences were within those contacted by the RNA polymerase during transcription initiation. Thus hypersensitivity is a characteristic of the RNA polymerase promoter complexes. The sequences needed for upmodulation included both upstream and downstream sequences in one case, only upstream sequences for another promoter and only downstream sequences for the third. Thus, there appear to be multiple mechanisms of transcription modulation by rifampicin.

Modulation of Salmonella gene expression by subinhibitory concentrations of quinolones.

Approximately 2.7% of a collection of Salmonella enterica var. Typhimurium promoter-lux reporter strains showed altered transcriptional patterns when exposed to low concentrations of nine different fluoroquinolones (FQs). Even at the subinhibitory concentrations employed, all nine FQs upregulated genes involved in the SOS response, umuD, lexA, sbmC and dinP. In addition, transcriptional regulators, genes putatively associated with membrane integrity (spr), virulence (sicA) and metabolism (plsB) were affected. Using the Ames test with Salmonella strain TA102, increased mutagenicity was demonstrated in response to all the FQs tested: ciprofloxacin, moxifloxacin, levofloxacin and gatifloxacin. Transcriptional effects were largely specific to the FQ antimicrobials. Such responses are consistent with the primary mechanism of action of this class of inhibitor, namely, the introduction of DNA damage. This work provides support for the notion that small molecules can have functions other than growth inhibition that may affect the establishment and maintenance of community dynamics in complex environments.

Improved lux reporters for use in Staphylococcus aureus.

The use of luxABCDE (lux) offers certain advantages over other reporters, such as: lacZ and xylE. It is real time and its signal generation is produced without the requirement for any additional substrates. In some bacteria such as Staphylococcus spp, light production by luciferase is restricted because of a limited availability of endogenous substrates such as fatty acid aldehyde. We describe the construction of promoterless-lux cloning vectors, pGYlux and pAmilux. S. aureus carrying B. subtilis xyl/tetO promoter fused to the lux genes of pGYlux gave up to a 2.5-fold enhancement of luminescence over S. aureus carrying the xyl/tetO promoter fused to lux genes of the previously published parent vector pAL2. Furthermore, pAmilux showed a 6-fold enhancement of lux expression when compared to pGYlux in S. aureus. This was achieved by cloning the constitutive ami promoter upstream of the luxCDE genes to increase endogenous fatty acid aldehyde production while maintaining its reporter functionality by fusing promoters to the luxAB genes.

Antibiotics as signalling molecules.

We present the argument that the majority of low-molecular-weight organic compounds made and secreted by microbes play roles as cell-signalling molecules in the environment. Of the large number of compounds isolated to date, only a small fraction have been shown to possess useful therapeutic antibiotic activity. However, most microbial metabolites modulate gene transcription at low concentrations, and this is proposed to be the primary effect of the compounds in the maintenance of microbial communities in the environment. Thus, microbial metabolites constitute a large collection of cell-signalling molecules that regulate gene expression in microbial populations and possibly the interactions of these populations with the surrounding organisms.

The world of subinhibitory antibiotic concentrations.

Although antibiotics have long been known to have multiple effects on bacterial cells at low concentrations, it is only with the advent of genome transcription analyses that these activities have been studied in detail at the level of cell metabolism. It has been shown that all antibiotics, regardless of their receptors and mode of action, exhibit the phenomenon of hormesis and provoke considerable transcription activation at low concentrations. These analyses should be of value in providing information on antibiotic side-effects, in bioactive natural product discovery and antibiotic mode-of-action studies.

Transcription modulation of Salmonella enterica serovar Typhimurium promoters by sub-MIC levels of rifampin.

Promoter-lux fusions that showed rifampin-modulated transcription were identified from a Salmonella enterica serovar Typhimurium 14028 reporter library. The transformation of a subset of fusions into mutants that lacked one of six global regulatory proteins or were rifampin resistant showed that transcription modulation was independent of the global regulators, promoter specific, and dependent on the interaction of rifampin with RNA polymerase.

The truth about antibiotics.

Microbes produce millions of organic compounds of low molecular weight--a world of very diverse chemical and biological ecology. We propose that, at the low concentrations likely to be found in the environment, the majority of these compounds play important roles in the modulation of metabolic function in natural microbial communities. The biological diversity is reflected by distinct target responses affecting a variety of transcription regulatory networks by different mechanisms. This provides the basis of chemical signalling processes in the microbial world and may well extend into many prokaryote-eukaryote interactions.

Dual effects of MLS antibiotics: transcriptional modulation and interactions on the ribosome.

The macrolide-lincosamide-streptogramin (MLS) antibiotics are an important group of translation inhibitors that act on the 50S ribosome. We show that, at subinhibitory concentrations, members of the MLS group modulate specific groups of bacterial promoters, as detected by screening a library of promoter-luxCDABE reporter clones of Salmonella enterica serovar Typhimurium. The patterns of transcription permit identification of classes of promoters having differential responses to antibiotics of related structure and mode-of-action; studies of antibiotic synergy or antagonism showed that eukaryotic translation inhibitors may act on the 50S ribosome. The mechanism of transcriptional modulation is not known but may involve bacterial stress responses and/or the disturbance and subsequent compensation of metabolic networks as a result of subtle interference with ribosome function. Transcriptional patterns detected with promoter-lux clones provide a novel approach to antibiotic discovery and mode-of-action studies.

Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics.

Antibiotics such as erythromycin and rifampicin, at low concentrations, alter global bacterial transcription patterns as measured by the stimulation or inhibition of a variety of promoter-lux reporter constructs in a Salmonella typhimurium library. Analysis of a 6,500-clone library indicated that as many as 5% of the promoters may be affected, comprising genes for a variety of functions, as well as a significant fraction of genes with no known function. Studies of a selection of the reporter clones showed that stimulation varied depending on the nature of the antibiotic, the promoter, and what culture medium was used; the response differed on solid as compared with liquid media. Transcription was markedly reduced in antibiotic-resistant hosts, but the presence of mutations deficient in stress responses such as SOS or universal stress did not prevent antibiotic-induced modulation. The results show that small molecules may have contrasting effects on bacteria depending on their concentration: either the modulation of bacterial metabolism by altering transcription patterns or the inhibition of growth by the inhibition of specific target functions. Both activities could play important roles in the regulation of microbial communities. These studies indicate that the detection of pharmaceutically useful natural product inhibitors could be effectively achieved by measuring activation of transcription at low concentrations in high-throughput assays using appropriate bacterial promoter-reporter constructs.