Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

ABSTRACTDNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of “poisons”, namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

The complete sequence, annotation and comparative analysis of the Escherichia coli IncFII family plasmid pMccB17, encoding biosynthetic and immunity functions for the antimicrobial peptide microcin B17

Microcin B17 (Mcb17) is a ribosomally synthesized and post-translationally modified peptide (RiPP), produced by Escherichia coli, that inhibits bacterial DNA gyrase in a similar way to quinolones. The Mcb17 operon, consisting of seven genes encoding biosynthetic and immunity/export functions, was originally found on a plasmid, pMccB17. This circular plasmid, previously known as pRYC17, was originally found in Escherichia coli strain LP17, isolated from the intestinal tract of a healthy newborn at Hospital La Paz, Spain and was transferred by conjugation to E. coli K-12 [Baquero et al. (1978) J. Bacteriol. 135: 342]. pMccB17 is a low copy number IncFII plasmid in the same incompatibility group as R100 and R1. Not much is known about this plasmid aside from the facts that it carries the Mcb17 operon, does not possess any conventional antibiotic resistance markers and its size was estimated to be approximately 70 kb. We extracted the plasmid from E. coli K-12 strain RYC1000 [pMccB17] and sequenced it twice using an Illumina short-read method, firstly together with the host bacterial chromosome, then plasmid DNA was purified and sequenced separately. PCR primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete sequence has 83 predicted genes, initially identified by Prokka and subsequently manually reannotated using BLAST. Comparison to other IncFII plasmids shows a large proportion of shared genes, especially in the conjugative plasmid backbone. However, pMccB17 which is a MOBF12 plasmid lacks transposable elements and in addition to the Mcb17 operon, this plasmid carries 25 genes of unknown function.

Protective Effect of Qnr on Agents Other than Quinolones That Target DNA Gyrase

ABSTRACTQnr is a plasmid-encoded and chromosomally determined protein that protects DNA gyrase and topoisomerase IV from inhibition by quinolones. Despite its prevalence worldwide and existence prior to the discovery of quinolones, its native function is not known. Other synthetic compounds and natural products also target bacterial topoisomerases. A number were studied as molecular probes to gain insight into how Qnr acts. Qnr blocked inhibition by synthetic compounds with somewhat quinolone-like structure that target the GyrA subunit, such as the 2-pyridone ABT-719, the quinazoline-2,4-dione PD 0305970, and the spiropyrimidinetrione pyrazinyl-alkynyl-tetrahydroquinoline (PAT), indicating that Qnr is not strictly quinolone specific, but Qnr did not protect against GyrA-targeting simocyclinone D8 despite evidence that both simocyclinone D8 and Qnr affect DNA binding to gyrase. Qnr did not affect the activity of tricyclic pyrimidoindole or pyrazolopyridones, synthetic inhibitors of the GyrB subunit, or nonsynthetic GyrB inhibitors, such as coumermycin A1, novobiocin, gyramide A, or microcin B17.Thus, in this set of compounds the protective activity of Qnr was confined to those that, like quinolones, trap gyrase on DNA in cleaved complexes.

Synthesis of full length and truncated microcin B17 analogues as DNA gyrase poisons

Using a combination of solid-phase peptide synthesis and fragment assembly strategies a library of full-length and truncated analogues of the antibacterial post-translationally modified peptide microcin B17 have been synthesised. Both antibacterial and DNA gyrase poisoning activities are also described for the synthetic analogues.

Unexpected consequences of administering bacteriocinogenic probiotic strains for Salmonella populations, revealed by an in vitro colonic model of the child gut

New biological strategies for the treatment of Salmonella infection are needed in response to the increase in antibiotic-resistant strains. Escherichia coli L1000 and Bifidobacterium thermophilum RBL67 were previously shown to produce antimicrobial proteinaceous compounds (microcin B17 and thermophilicin B67, respectively) active in vitro against a panel of Salmonella strains recently isolated from clinical cases in Switzerland. In this study, two three-stage intestinal continuous fermentation models of Salmonella colonization inoculated with immobilized faeces of a two-year-old child were implemented to study the effects of the two bacteriocinogenic strains compared with a bacteriocin-negative mutant of strain L1000 on Salmonella growth, as well as gut microbiota composition and metabolic activity. Immobilized E. coli L1000 added to the proximal colon reactor showed a low colonization, and developed preferentially in the distal colon reactor independent of the presence of genetic determinants for microcin B17 production. Surprisingly, E. coli L1000 addition strongly stimulated Salmonella growth in all three reactors. In contrast, B. thermophilum RBL67 added in a second phase stabilized at high levels in all reactors, but could not inhibit Salmonella already present at a high level (>107 c.f.u. ml−1) when the probiotic was added. Inulin added at the end of fermentation induced a strong bifidogenic effect in all three colon reactors and a significant increase of Salmonella counts in the distal colon reactor. Our data show that under the simulated child colonic conditions, the microcin B17 production phenotype does not correlate with inhibition of Salmonella but leads to a better colonization of E. coli L1000 in the distal colon reactor. We conclude that in vitro models with complex and complete gut microbiota are required to accurately assess the potential and efficacy of probiotics with respect to Salmonella colonization in the gut.