Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

Schmutz, Elisabeth; Hennig, Susanne; Li, Shu-Ming; Heide, Lutz

doi:10.1099/mic.0.26867-0

Cited by 28 publications

(26 citation statements)

References 22 publications

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“…Sequence analysis of the biosynthetic gene cluster of simocyclinone revealed that it contains several genes with high degrees of similarity to those involved in the biosynthesis of the coumarin moieties of novobiocin, clorobiocin, and coumermycin (13,39). However, the simocyclinone gene cluster does not contain an obvious resistance gene that would give a clue as to its target (13,39); this is in contrast to the biosynthetic gene clusters of novobiocin, clorobiocin, and coumermycin, all of which contain genes encoding aminocoumarin-resistant topoisomerase subunits (35,36). Simocyclinone D8 has been shown to exhibit antibiotic activity against gram-positive bacteria, as well as cytostatic effects against human tumor cell lines; little or no activity has been found against gram-negative organisms such as E. coli (32,33).…”

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confidence: 52%

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

Flatman

Howells

Heide³

et al. 2005

Antimicrob Agents Chemother

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109

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We have characterized the interaction of a new class of antibiotics, simocyclinones, with bacterial DNA gyrase. Even though their structures include an aminocoumarin moiety, a key feature of novobiocin, coumermycin A 1 , and clorobiocin, which also target gyrase, simocyclinones behave strikingly differently from these compounds. Simocyclinone D8 is a potent inhibitor of gyrase supercoiling, with a 50% inhibitory concentration lower than that of novobiocin. However, it does not competitively inhibit the DNA-independent ATPase reaction of GyrB, which is characteristic of other aminocoumarins. Simocyclinone D8 also inhibits DNA relaxation by gyrase but does not stimulate cleavage complex formation, unlike quinolones, the other major class of gyrase inhibitors; instead, it abrogates both Ca 2؉ -and quinolone-induced cleavage complex formation. Binding studies suggest that simocyclinone D8 interacts with the N-terminal domain of GyrA. Taken together, our results demonstrate that simocyclinones inhibit an early step of the gyrase catalytic cycle by preventing binding of the enzyme to DNA. This is a novel mechanism for a gyrase inhibitor and presents new possibilities for antibacterial drug development.DNA topoisomerases are enzymes that control the topology of DNA in cells (5). The enzymes are divided into two types, topoisomerases I and II, depending on whether their reactions involve breakage of one or both strands of the DNA. Overall, the mechanism of action of topoisomerases involves the passage of one segment of DNA through a break in another. This reaction can achieve all the known reactions of topoisomerases: relaxation and supercoiling, catenation and decatenation, and knotting and unknotting. DNA supercoiling activity is unique to gyrase, and as this enzyme is essential in all bacteria but is not found in humans, it is an ideal target for antibacterials (24). The enzyme consists of an A 2 B 2 heterotetramer. The A subunit (GyrA; 97 kDa in Escherichia coli) comprises an N-terminal domain involved in DNA cleavage and religation and a C-terminal DNA-wrapping domain. The B subunit (GyrB; 90 kDa in E. coli) contains the site of ATP hydrolysis at the N-terminal domain (GyrB43); the C-terminal domain interacts with both GyrA and DNA. To introduce supercoils, the enzyme wraps a double-stranded segment around itself, cleaves this DNA (the gate segment) in both strands, passes the wrapped DNA (transported segment) through the break, and then reseals the DNA.DNA gyrase is a proven target for antibacterial agents (24). Two well-studied classes of drugs target the enzyme: the coumarins and the quinolones. Quinolones are synthetic compounds that are widely used in clinical practice (8). Coumarins are naturally occurring products of Streptomyces species which, despite being more potent inhibitors of gyrase in vitro than quinolones, have had limited clinical use due to their low solubility, poor uptake, and eukaryotic cell toxicity (27). Other gyrase inhibitors include the cyclothialidines and the bacterial toxins CcdB and m...

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confidence: 52%

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

Flatman

Howells

Heide³

et al. 2005

Antimicrob Agents Chemother

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109

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“…Apparently, this antibiotic has evolved for optimal interaction with its targets, gyrase and topo IV. In this context, it is noteworthy that the clorobiocin gene cluster contains two resistance genes protecting the natural clorobiocin producer Streptomyces roseochromogenes from the toxic effect of the antibiotic: gyrB R , which encodes an aminocoumarin-resistant gyrase subunit, and parY R , which encodes an aminocoumarin-resistant topo IV subunit (20). In contrast, the novobiocin biosynthetic gene cluster contains only a gyrB R resistance gene (21,22).…”

Section: Resultsmentioning

confidence: 99%

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

Flatman

Eustáquio²,

Li³

et al. 2006

Antimicrob Agents Chemother

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Novobiocin and clorobiocin are gyrase inhibitors produced by Streptomyces strains. Structurally, the two compounds differ only by substitution at two positions: CH 3 versus Cl at position 8 of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3؆-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH 3 , or Cl at 8 and H, carbamoyl, or MePC at 3؆-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3؆-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8 further enhanced activity, but the effect of CH 3 or Cl depended on the nature of the acyl group at 3؆: in the presence of carbamoyl at 3؆, CH 3 resulted in higher activity; in the presence of MePC at 3؆, Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4؆-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.

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“…Streptomycetes, however, have no xerCD-like genes (6, 23), though they do have a topoisomerase IV biochemically able to catalyze ATP-dependent decatenation (6,23,38). Whether this enzyme plays a role in chromosome decatenation during sporulation and/or interacts with FtsK SC is yet to be revealed.…”

Section: Discussionmentioning

confidence: 99%

Role of an FtsK-Like Protein in Genetic Stability inStreptomyces coelicolorA3(2)

Wang

et al. 2007

J Bacteriol

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Streptomyces coelicolor A3(2) does not have a canonical cell division cycle during most of its complex life cycle, yet it contains a gene (ftsK SC ) encoding a protein similar to FtsK, which couples the completion of cell division and chromosome segregation in unicellular bacteria such as Escherichia coli. Here, we show that various constructed ftsK SC mutants all grew apparently normally and sporulated but upon restreaking gave rise to many aberrant colonies and to high frequencies of chloramphenicol-sensitive mutants, a phenotype previously associated with large terminal deletions from the linear chromosome. Indeed, most of the aberrant colonies had lost large fragments near one or both chromosomal termini, as if chromosome ends had failed to reach their prespore destination before the closure of sporulation septa. A constructed FtsK SC -enhanced green fluorescent protein fusion protein was particularly abundant in aerial hyphae, forming distinctive complexes before localizing to each sporulation septum, suggesting a role for FtsK SC in chromosome segregation during sporulation. Use of a fluorescent reporter showed that when ftsK SC was deleted, several spore compartments in most spore chains failed to express the late-sporulation-specific sigma factor gene sigF, even though they contained chromosomal DNA. This suggested that sigF expression is autonomously activated in each spore compartment in response to completion of chromosome transfer, which would be a previously unknown checkpoint for late-sporulation-specific gene expression. These results provide new insight into the genetic instability prevalent among streptomycetes, including those used in the industrial production of antibiotics.Bacteria of the gram-positive genus Streptomyces are of great interest for their mycelial growth and complex development, as well as being major producers of antibiotics. On solid media, Streptomyces spores germinate and grow into a vegetative mycelium from which aerial hyphae emerge, eventually developing into chains of unigenomic spores. Vegetative hyphae have only sparse septation without actual cell separation; each hyphal compartment contains several unsegregated copies of the chromosome (16). In contrast, during sporulation of aerial hyphae, regularly spaced septa form synchronously in long multigenomic compartments, each receiving one chromosome, and cell separation eventually takes place. Unlike the situation in dividing bacteria such as Escherichia coli, initiation of sporulation septation in Streptomyces often occurs over unsegregated chromosomes, which do not separate until very late in septation (31, 39). This raises the question of how Streptomyces clears chromosomal DNA from sites of septal closure to ensure faithful segregation of the replicated linear chromosomes to progeny spores, avoiding guillotining by septal closure. Completion of chromosome transfer to daughter cells in E. coli is carried out by a septum-located multifunctional protein, FtsK, which is deposited at the FtsZ ring and couples closure of t...

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Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

Cited by 28 publications

References 22 publications

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

Simocyclinone D8, an Inhibitor of DNA Gyrase with a Novel Mode of Action

Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis

Role of an FtsK-Like Protein in Genetic Stability inStreptomyces coelicolorA3(2)

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