High-throughput screen reveals sRNAs regulating crRNA biogenesis by targeting CRISPR leader to repress Rho termination.

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.

Genome-wide relationship between R-loop formation and antisense transcription in Escherichia coli.

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA-DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.

Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma.

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.

Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M.

The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho) inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho's structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO) as a template in MODELLER (v 9.10). The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9) and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial) database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds - ZINC24934545 and ZINC72319544 - that showed high binding affinity among 2,829 drug analogs that bind with key active-site residues; these residues are considered for protein-ligand binding and unbinding pathways via steered molecular dynamics simulations. Arg215 in the model plays an important role in the stability of the protein-ligand complex via a hydrogen bonding interaction by aromatic-π contacts, and the ADMET (absorption, distribution, metabolism, and excretion) analysis of best leads indicate nontoxic in nature with good potential for drug development.

Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum.

Riboswitches are RNA elements that regulate gene expression in response to their ligand. Although these regulations are thought to be performed without any aid of other factors, recent studies suggested the participation of protein factors such as transcriptional termination factor Rho and RNase in some riboswitch regulations. However, to what extent these protein factors contribute to the regulation was unclear. Here, we studied the regulatory mechanism of the flavin mononucleotide (FMN) riboswitch of Corynebacterium glutamicum which controls the expression of downstream ribM gene. Our results showed that this riboswitch downregulates both ribM mRNA and RibM protein levels in FMN-rich cells. Analysis of mRNA stability and chromatin immunoprecipitation-real-time PCR analysis targeting RNA polymerase suggested the involvement of the mRNA degradation and premature transcriptional termination in this regulation, respectively. Simultaneous disruption of RNase E/G and Rho function completely abolished the regulation at the mRNA level. Also, the regulation at the protein level was largely diminished. However, some FMN-dependent regulation at the protein level remained, suggesting the presence of other minor regulatory mechanisms. Altogether, we demonstrated for the first time that two protein factors, Rho and RNase E/G, play a central role in the riboswitch-mediated gene expression control.

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

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.

Deficiency of autotaxin/lysophospholipase D results in head cavity formation in mouse embryos through the LPA receptor-Rho-ROCK pathway.

Autotaxin, encoded by the Enpp2 gene, generates lysophosphatidic acid (LPA) extracellularly, eliciting various cellular responses through specific LPA receptors. Previous studies have revealed that Enpp2(-/-) mice die at E9.5 owing to angiogenic defects in the yolk sac. Moreover, Enpp2(-/-) embryos show growth retardation, allantois malformation, no axial turning, and head cavity formation. We have also demonstrated that lysosome biogenesis is impaired in yolk sac visceral endoderm cells of Enpp2(-/-) embryos as a result of the downregulation of the Rho-ROCK (Rho-associated coiled-coil containing protein kinase)-LIM kinase pathway. In this study, we examine what signaling defect(s) is responsible for head cavity formation and yolk sac angiogenic defects. By using a whole embryo culture system, we show that 10 µM Ki16425, an antagonist for the LPA receptors, induces head cavity formation and yolk sac angiogenic defects in wild-type embryos. Moreover, 1 µM Ki16425 induces both phenotypes in Enpp2 heterozygous embryos at significantly higher incidence than in wild-type embryos, suggesting an interaction between autotaxin and LPA receptor signaling. Furthermore, we show that inhibition of the Rho-ROCK pathway induces head cavity formation, whereas multiple pathways are involved in yolk sac angiogenic defects. These results reveal the signal transduction defects that underlie the abnormalities in Enpp2(-/-) embryos.

Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli.

Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.

Crucial role of Rho-nuclear factor-kappaB axis in angiotensin II-induced renal injury.

This study was performed to determine the effectiveness of the Rho kinase inhibitor and NF-kappaB inhibitor in renal injury of ANG II-infused hypertensive rats. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation (n = 7) or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG II-infused rats were further subdivided into three subgroups (n = 7 each) to receive one of the following treatments during the entire period: vehicle, Rho kinase inhibitor (fasudil; 3 mg.kg(-1).day(-1) ip), or NF-kappaB inhibitor (parthenolide; 1 mg.kg(-1).day(-1) ip). After 12 days of ANG II infusion, systolic blood pressure (BP; 208 +/- 7 vs. 136 +/- 3 mmHg), Rho kinase activity, NF-kappaB activity, renal ANG II contents (160 +/- 25 vs. 84 +/- 14 pg/g), monocytic chemotactic protein (MCP) 1 mRNA, interstitial macrophage infiltration, transforming growth factor-beta1 (TGF-beta1) mRNA, interstitial collagen-positive area, urinary protein excretion (43 +/- 6 vs. 11 +/- 2 mg/day), and urinary albumin excretion were significantly enhanced compared with the Sham group. While fasudil or parthenolide did not alter systolic BP (222 +/- and 190 +/- 21, respectively), both treatments completely blocked ANG II-induced enhancement of NF-kappaB activity, renal ANG II contents (103 +/- 11 and 116 +/- 21 pg/g, respectively), MCP1 mRNA, interstitial macrophage infiltration, TGF-beta1 mRNA, interstitial collagen-positive area, urinary protein excretion (28 +/- 6 and 23 +/- 3 mg/day, respectively), and urinary albumin excretion. Importantly, parthenolide did not alter ANG II-induced Rho kinase activation although fasudil abolished ANG II-induced Rho kinase activation. These data indicate that the Rho-NF-kappaB axis plays crucial roles in the development of ANG II-induced renal injury independently from BP regulation.

The molecular basis for the mode of action of bicyclomycin.

Bicyclomycin (1) is a clinically useful antibiotic exhibiting activity against a broad spectrum of Gram-negative bacteria and against the Gram-positive bacterium, Micrococcus luteus. Bicyclomycin has been used to treat diarrhea in humans and bacterial diarrhea in calves and pigs and is marketed by Fujisawa (Osaka, Japan) under the trade name Bicozamycin. The structure of 1 is unique among antibiotics, and our studies document that its mechanism of action is novel. Early mechanistic proposals suggested that 1 reacted with nucleophiles (e.g., a protein sulfhydryl group) necessary for the remodeling the peptidoglycan assembly within the bacterial cell wall. We, however, showed that 1 targeted the rho transcription termination factor in Escherichia coli. The rho protein is integral to the expression of many gene products in E. coli and other Gram-negative bacteria, and without rho the cell losses viability. Rho is a member of the RecA-type ATPase class of enzymes that use nucleotide contacts to couple oligonucleotide translocation to ATP hydrolysis. Bicyclomycin is the only known selective inhibitor of rho. In this article, we integrate the evidence obtained from bicyclomycin structure-activity studies, site-directed mutagenesis investigations, bicyclomycin affinity labels, and biochemical and biophysical measurements with recent X-ray crystallographic images of the bicyclomycin-rho complex to define the rho antibiotic binding site and to document the pathway for rho inhibition by 1. Together, the structural and functional studies demonstrate how 1, a modest rho inhibitor, can disrupt the rho molecular machinery thereby leading to a catastrophic effect caused by the untimely overproduction of proteins not normally expressed constitutively, thus leading to a toxic effect on the cells.

Bismuth-dithiol inhibition of the Escherichia coli rho transcription termination factor.

Bismuth-dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth-dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay (I50=60 microM) and as a competitive inhibitor with respect to ribo(C)10 in the poly(dC)-ribo(C)10-dependent ATPase assay. The minimum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 microM. Using the tnaA/lacZ fusion reporter assay we showed that sublethal amounts (3 microM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo beta-galactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy.

Galphaz inhibits serum response factor-dependent transcription by inhibiting Rho signaling.

Galpha12/13 or Galphaq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Galphaz signals inhibit serum response factor (SRF)-dependent transcription. Galphaz expression inhibits Galpha12/13-, Galphaq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Galphaz mutants with defective fatty acylation has no inhibitory effect. Expression of Galphaz, but not Galphai, attenuates serum-induced SRE reporter activation, suggesting that Galphaz can down-regulate endogenous signals leading to SRF. Whereas Galphaz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect Galpha12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Galphaz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Galphaz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Galphaz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Galphaz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.

Metal-1,4-dithio-2,3-dihydroxybutane chelates: novel inhibitors of the Rho transcription termination factor.

Rho is an enzyme that is essential for the growth and survival of Escherichia coli, and bicyclomycin (1) is its only known selective inhibitor. We show that metal (Cd(2+), Ni(2+), and Zn(2+)) complexes of 1,4-dithio-2,3-dihydroxybutanes (2) serve as effective and potent rho inhibitors with I(50) values that can exceed that of 1. Maximal inhibition for ZnCl(2) and L-dithiothreitol (2a) corresponded to Zn(2):L-DTT stoichiometry. The I(50) value for the 2:1 Zn-L-DTT solution was 20 microM, which made it 3 times more potent than 1 (I(50) = 60 microM). Kinetic studies showed that a Zn-L-DTT solution functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay and as a competitive inhibitor with respect to ribo(C)(10) in the poly(dC).ribo(C)(10)-stimulated ATPase assay. These findings demonstrated that both 1 and a Zn-L-DTT solution disrupted rho-mediated ATP hydrolysis but that they inhibit using different mechanisms. Substitution of L-DTT with 1,2-ethanedithiol in ZnCl(2) solutions led to a comparable loss of rho poly(C)-dependent ATPase activity, indicating that other metal chelates can serve as efficient inhibitors. The site and pathway of rho inhibition by the putative metal-1,4-dithio-2,3-dihydroxybutane chelates are discussed in light of the current data.

Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties.

Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1.rho interaction-K(d), stoichiometry for 1.rho binding, and whether 1 and ATP binding induce conformational changes in rho-remain unknown. In this study, the design, synthesis, and characterization of a series of bicyclomycin fluorescent probes (BFP) constructed to sense the 1.rho interaction are described and their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay. The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave a K(d) (15) of 9.9 microM. Using ADP in place of ATP or excluding nucleotide did not result in energy transfer, which suggests that ATP binding induced a conformational change in rho. FRET measurements provided an approximate weighted average distance (23 A) between W381 and 15 in the presence of bound ATP. The K(d) value for 15.rho was correlated with ATP binding at the 3 tight ATP binding (K(d)(ATP) = 95 nM) sites in wild-type rho.

A new inhibitor of the transcription-termination factor Rho.

In this study we describe BI-K0058, a new inhibitor of the transcription-termination factor Rho belonging to a different chemical class from bicyclomycin, the only known antibiotic acting on Rho. BI-K0058 inhibits the poly(C)-dependent ATPase activity of Rho with an IC(50) of 25 microM as well as in vitro transcription-termination of two natural substrates, the Salmonella enterica hisG cistron and the f1 phage intergenic region. BI-K0058 does not affect photolabeling of Rho by ATP. The results of gel mobility shift experiments with a natural RNA substrate demonstrate that BI-K0058 inhibits the formation of the ATP-independent high affinity Rho-RNA complex.

The Mg2+ requirements for rho transcription termination factor: catalysis and bicyclomycin inhibition.

Kinetic studies document that the essential Escherichia coli transcription termination factor rho utilizes Mg(2+) and ATP as a substrate and requires a second Mg(2+) ion for maximum poly(C)-dependent ATP hydrolysis activity. The velocity curves show a classic nonessential Mg(2+) activation pattern in which Mg(2+) augments hydrolysis by 39% and gives a K(1)' for MgATP of 9.5 microM in the presence of excess Mg(2+) and a K(1) for MgATP of 21.2 microM under limiting Mg(2+) concentrations. Bicyclomycin (1), a commercial antibiotic that inhibits rho, weakened Mg(2+) binding at the nonessential site and disrupted the nonessential Mg(2+) activation pathway for poly(C)-dependent ATP hydrolysis. The K(i) values for 1 were 23 microM and 35 microM under excess and limiting Mg(2+) conditions, respectively, while the K(Mg(app)) for nonessential Mg(2+) increased with increasing 1 concentrations. These findings, when combined with reported mechanistic studies, provide an emerging picture of key catalytic and substrate binding sites that are necessary for rho function and that are proximal to the 1 binding site.

C(5)-C(5a)-modified bicyclomycins: synthesis, structure, and biochemical and biological properties.

Bicyclomycin (1) is a novel antibiotic that targets rho transcription termination factor in Escherichia coli. We have demonstrated that retention of the C(5)-C(5a) exomethylene unit in 1 is not essential for inhibition. In a recent paper we proposed a working model for 1 and rho function and suggested that 1 binds in a cleft with the C(5)-C(5a) exomethylene unit directed toward the dimeric interface of two rho monomers. This report examines the bicyclomycin C(5)-C(5a) structural constraints necessary for retention of rho inhibitory activity. Three classes of C(5)-C(5a)-modified bicyclomycins have been prepared and their inhibitory activities evaluated in the poly C-dependent ATPase and filter disk antimicrobial assays. The first series consisted of 12 analogues (8-19) that contained a C(5a)-unsaturated substituent and possessed C(5E)-geometry. The second set were a pair of C(5a)-substituted C(5E)- and C(5Z)-geometrical isomers (21 and 23). The final group of compounds consisted of six C(5)-C(5a)-dihydrobicyclomycins (24-28, 34) where the terminal substituent was systematically varied. We find that extending the C(5)-C(5a) double bond with unsaturated substituents provides bicyclomycin derivatives with excellent inhibitory activities in the biochemical assay, and that enhanced inhibitory activity is observed for the C(5E) geometrical isomer compared with its C(5Z) counterpart. Finally, C(5a)-substituted dihydrobicyclomycin inhibitory activity appears to be tightly regulated by the nature and spatial placement of the C(5a)-terminal substituent with respect to the [4.2.2]-bicyclic ring system. The observed biochemical activities for the C(5a)-extended conjugated bicyclomycin derivatives and the (5E) and (5Z) isomers were correlated with a structural model for the 1-rho complex.