Bicyclomycin Research Articles

Bicyclomycin generates ROS and blocks cell division in Escherichia coli.

The role of reactive oxygen species (ROS) in the killing exerted by antibiotics on bacteria is debated. Evidence attributes part of toxicity of many antibiotics to their ability to generate ROS by interfering with cellular metabolism, but some studies dismiss the role of ROS. Bicyclomycin (BCM) is a broad-spectrum antibiotic that is the only known compound to inhibit E. coli transcription terminator factor Rho with no known other cellular targets. In the present study, we addressed this question by checking whether the induction of oxidative stress could explain the increased sensitivity to Bicyclomycin in the hns deleted strain even in Δkil background in E. coli. BCM evoked the generation of ROS in E. coli cells. BCM is known to cause the cell filamentation phenotype in E. coli. Performing fluorescence microscopic analysis, we show that bicyclomycin-dependent cell filamentation is associated with SOS response. RecA-GFP filaments were found to colocalize with the damaged DNA sites in the cell. Further analysis revealed that the genomic DNA was partitioned but the cell septum formation was severely affected under BCM treatment. Furthermore, we observed biofilm formation by E. coli after BCM treatment. We hypothesize that ROS production after BCM treatment could lead to cell filamentation in bacteria. A better understanding of the mode of toxicity of BCM will help us design better antibiotic treatment regimes for clinical practices, including combinatorial drug therapies. The cell filamentation phenotype observed after BCM treatment makes this antibiotic a promising drug for phage-antibiotic synergy (PAS) therapy.

Continuous bioactivity-dependent evolution of an antibiotic biosynthetic pathway

Antibiotic biosynthetic gene clusters (BGCs) produce bioactive metabolites that impart a fitness advantage to their producer, providing a mechanism for natural selection. This selection drives antibiotic evolution and adapts BGCs for expression in different organisms, potentially providing clues to improve heterologous expression of antibiotics. Here, we use phage-assisted continuous evolution (PACE) to achieve bioactivity-dependent adaptation of the BGC for the antibiotic bicyclomycin (BCM), facilitating improved production in a heterologous host. This proof-of-principle study demonstrates that features of natural bioactivity-dependent evolution can be engineered to access unforeseen routes of improving metabolic pathways and product yields.

A Six-Oxidase Cascade for Tandem C-H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis.

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe2+ -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.

A Six‐Oxidase Cascade for Tandem C−H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis

AbstractAs a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O‐bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo‐methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one‐pot enzymatic synthesis. A tRNA‐dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α‐ketoglutarate/Fe2+‐dependent dioxygenases and one cytochrome P450 monooxygenase, regio‐ and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo‐methylene moiety, and a medium‐sized bridged ring through the functionalization of eight unactivated C−H bonds.

A Screen for rfaH Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho.

ABSTRACTRfaH activates horizontally acquired operons that encode lipopolysaccharide core components, pili, toxins, and capsules. Unlike its paralog NusG, which potentiates Rho-mediated silencing, RfaH strongly inhibits Rho. RfaH is recruited to its target operons via a network of contacts with an elongating RNA polymerase (RNAP) and a specific DNA element called ops to modify RNAP into a pause- and NusG-resistant state. rfaH null mutations confer hypersensitivity to antibiotics and detergents, altered susceptibility to bacteriophages, and defects in virulence. Here, we carried out a selection for suppressors that restore the ability of a ΔrfaH mutant Escherichia coli strain to grow in the presence of sodium dodecyl sulfate. We isolated rho, rpoC, and hns suppressor mutants with changes in regions previously shown to be important for their function. In addition, we identified mutants with changes in an unstructured region that connects the primary RNA-binding and helicase domains of Rho. The connector mutants display strong defects in vivo, consistent with their ability to compensate for the loss of RfaH, and act synergistically with bicyclomycin (BCM), which has been recently shown to inhibit Rho transformation into a translocation-competent state. We hypothesize that the flexible connector permits the reorientation of Rho domains and serves as a target for factors that control the motor function of Rho allosterically. Our results, together with the existing data, support a model in which the connector segment plays a hitherto overlooked role in the regulation of Rho-dependent termination.

Single-Gene Deletion Mutants of Escherichia coli with Altered Sensitivity to Bicyclomycin, an Inhibitor of Transcription Termination Factor Rho

We have screened the entire KEIO collection of 3,985 single-gene knockouts in Escherichia coli for increased susceptibility or resistance to the antibiotic bicyclomycin (BCM), a potent inhibitor of the transcription termination factor Rho. We also compared the results to those of a recent study we conducted with a large set of antibiotics (A. Liu et al., Antimicrob. Agents Chemother. 54:1393-1403, 2010). We find that deletions of many different types of genes increase sensitivity to BCM. Some of these are involved in multidrug sensitivity/resistance, whereas others are specific for BCM. Mutations in a number of DNA recombination and repair genes increase BCM sensitivity, indicating that DNA damage leading to single- and double-strand breaks is a downstream effect of Rho inhibition. MDS42, which is deleted for all cryptic prophages and insertion elements (G. Posfai et al., Science 312:1044-1046, 2006), or W3102 deleted for the rac prophage-encoded kil gene, are partially resistant to BCM (C. J. Cardinale et al., Science 230:935-938, 2008). Deletion of cryptic prophages also overcomes the increased BCM sensitivity in some but not all mutants examined here. Deletion of the hns gene renders the cell more sensitive to BCM even in the Δkil or MDS42 background. This suggests that BCM activates additional modes of cell death independent of Kil and that these could provide a target to potentiate BCM killing.

Rho directs widespread termination of intragenic and stable RNA transcription

The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found approximately 200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation.

Patterns of resistance to florfenicol and bicyclomycin in Brazilian strains of motile aeromonads

The minimum inhibitory concentration (MIC) and diameter of zone of inhibition of florfenicol (FLO) and the MIC of bicyclomycin (BCM) were determined for 118 motile aeromonad strains isolated from diseased fish and pond water in Brazil. Tests were performed according to Clinical and Laboratory Standards Institute guidelines. FLO MIC values ranged from 0.5 to 16 μg/mL and BCM MIC values from 0.78 to 100 μg/mL. According to MIC frequency distributions, the strains were classified as wild-type (WT) and non-wild-type (NWT) and provisional epidemiological cutoff values for FLO (WT ≤ 4 μg/mL) and BCM (WT ≤ 6.25 μg/mL) were estimated. Disk susceptibility tests revealed a provisional epidemiological cutoff of WT ≥ 23 mm for FLO. The lack of groups of bacterial strains with NWT behavior to FLO and BCM suggests that the estimated cutoffs should be revised in the future. Both antibiotics exhibited high efficacy in vitro against motile aeromonad strains.

Structural Mechanism of Inhibition of the Rho Transcription Termination Factor by the Antibiotic Bicyclomycin

Rho is a hexameric RNA/DNA helicase/translocase that terminates transcription of select genes in bacteria. The naturally occurring antibiotic, bicyclomycin (BCM), acts as a noncompetitive inhibitor of ATP turnover to disrupt this process. We have determined three independent X-ray crystal structures of Rho complexed with BCM and two semisynthetic derivatives, 5a-(3-formylphenylsulfanyl)-dihydrobicyclomycin (FPDB) and 5a-formylbicyclomycin (FB) to 3.15, 3.05, and 3.15 A resolution, respectively. The structures show that BCM and its derivatives are nonnucleotide inhibitors that interact with Rho at a pocket adjacent to the ATP and RNA binding sites in the C-terminal half of the protein. BCM association prevents ATP turnover by an unexpected mechanism, occluding the binding of the nucleophilic water molecule required for ATP hydrolysis. Our data explain why only certain elements of BCM have been amenable to modification and serve as a template for the design of new inhibitors.

Identifying the Bicyclomycin Binding Domain through Biochemical Analysis of Antibiotic-resistant Rho Proteins

Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket.

Studies on the reactivity of bicyclomycin 3'-O-methanesulfonate. A novel ring-expansion transformation

Studies on the reactivity of bicyclomycin 3'-O-methanesulfonate. A novel ring-expansion transformation

Observations on the activation of bicyclomycin

Observations on the activation of bicyclomycin

ChemInform Abstract: THE CHIRAL SYNTHESIS OF BICYCLOMYCIN

AbstractDie Synthese von optisch aktivem Bicyclomycin (III) erfolgt in 17 Stufen.

THE CHIRAL SYNTHESIS OF BICYCLOMYCIN

Abstract The optically active bicyclomycin was successfully synthesized in 17 steps from N,N′-diacetyl-anhydroglycine.

Antigenicity of bicyclomycin and its cross-reactivity with benzylpenicillin.

Antigenicity of bicyclomycin and its cross-reactivity with benzylpenicillin.