By ser esteraseprotease inhibitors figure s2

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by Ser esterase/protease inhibitors (Figure S2), consistent with the possible involvement of a metallo-esterase. This contrasts with our recent analysis of Streptomyces daptomycin esterases, which appear to use canonical Ser catalytic triad chemistry to inactivate the antibiotic [37]. Furthermore, unlike inactivation by Streptomyces , which appears to be constitutively expressed, the production of the inactivating activity in P. lautus was inducible by exposure to daptomycin (Figure 5). To explore if this activity was unique to the cave isolate, we obtained a surface strain of P. lautus (ATCC 43898) and observed similar results in enzymatic inactivation and antibiotic-associated induction of activity. Antibiotic Resistance in Cave Bacteria PLoS ONE | 3 April 2012 | Volume 7 | Issue 4 | e34953
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Antibiotic Inactivation by Gram-negative isolates We observed little enzyme-mediated antibiotic inactivation in Gram-negative isolates (Table 2), suggesting that there are other molecular mechanisms of resistance at play such as efflux, target modification, or barriers to entry. Three strains belonging to the genera Agrobacterium and Ochoromobactrum displayed chloramphen- icol inactivation, which we determined by LC/MS to be the modification by acetylation (Table S5); a well-established resis- tance mechanism both in Gram-positive and Gram-negative bacteria [33]. Characterization of a B. paraconglomeratum macrolide kinase Phosphorylation of macrolides in pathogenic bacteria is a growing clinical problem [38] catalyzed by a family of macrolide phosphotransferases (MPHs) [36,39]. In order to probe for the presence of possible mph genes in macrolide inactivating B. paraconglomeratum , we prepared a draft genome sequence using Roche 454 and Illumina platforms. A gene encoding a predicted macrolide kinase, mphE , was identified (Figure 6). We expressed the gene product in E. coli , purified the enzyme and determined its activity and specificity using steady state kinetics (Table 3). The enzyme efficiently modified 14-, 15-, and 16-membered macrolide antibiotics aligning this enzyme with known type II MPHs (based on previous characterization of these enzymes in E. coli isolates) [40,41]. The regiospecificity of phosphorylation of telithromycin was determined by multidimensional and multinuclear magnetic resonance analysis to be at the 2 9 -hydroxyl group of the desosamine sugar (Figures S3 and S4, Tables S6 and S7). The genome of another Brachybacterium species has been reported, Brachybacterium faecium DSM 4810, a terrestrial soil isolate Figure 2. Resistance levels of Lechuguilla cave bacteria at 20 m g/ml against various antibiotics: (top) Gram-positive strains (bottom) Gram-negative strains. Antibiotics are grouped according to their mode of action/target, where each color represents a different target. doi:10.1371/journal.pone.0034953.g002 Antibiotic Resistance in Cave Bacteria PLoS ONE | 4 April 2012 | Volume 7 | Issue 4 | e34953
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[42]. The B. faecium genome includes a putatively annotated aminoglycoside kinase, that is 72% identical at the amino acid level to the B. paraconglomeratum MPH. Expression, purification and analysis of the B. faecium enzyme
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