Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae

Mosher, Roy H.; Ranade, Neelima; Schrempf, Hildgund; Vining, L. C.

doi:10.1099/00221287-136-2-293

Cited by 38 publications

(25 citation statements)

References 27 publications

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“…In addition, the macrolide esterase and a fosfomycin-specific epoxide hydrolase were also shown to mediate the related drug resistance (10,12,23). The Cm hydrolase activity has been reported in the Cm-producing actinomycetes; however, its exact mechanism remains unclear (16,19). This is in contrast to CAT and drug efflux pumps, which are widespread in numerous bacteria and are well characterized (25).…”

Section: Discussionmentioning

confidence: 59%

Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

Wang

Lee

et al. 2012

Appl Environ Microbiol

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ABSTRACTChloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts ofEscherichia coliexpressing a chloramphenicol acetate esterase gene,estDL136. A hydrolysate of chloramphenicol was identified asp-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. WhenestDL136was expressed inE. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol onE. coli, due to their inactivation. In addition,E. colicarryingestDL136deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flankingestDL136encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial familySphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster withestDL136inE. coliis involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

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Section: Discussionmentioning

confidence: 59%

Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

Wang

Lee

et al. 2012

Appl Environ Microbiol

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“…Twelve of them are clustered on the approximately 18 kb of chromosomal DNA sequenced in this and previous studies (Mosher et al, 1990(Mosher et al, , 1995Brown et al, 1996;Chang et al, 2001;He et al, 2001). Functions for nine genes have been confirmed by insertional inactivation, and particular interest has focused on one (cmlP; see Fig.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

2004

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Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1 H-NMR established that the principal metabolite in the disrupted strains was the a-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH 2 -dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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“…For S. aurantia and other bacteria with potent esterase activity, selection using resistance mechanisms that are not based on acetylation may be necessary. In the case of streptomycetes and chloramphenicol, these other mechanisms include hydrolases (17,22), phosphorylases (21), and extrusion pumps (10).…”

Section: Discussionmentioning

confidence: 99%

Spirochaeta aurantia Has Diacetyl Chloramphenicol Esterase Activity

Sohaskey

Barbour

2000

J Bacteriol

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The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, ␣-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.The antibiotic chloramphenicol blocks translation by interacting with the peptidyl transferase centers of ribosomes (25). Acquired resistance to chloramphenicol in eubacteria is most commonly provided by the enzyme chloramphenicol acetyltransferase (CAT), which is encoded by one of several different types of cat genes (reviewed in reference 27). Some CAT proteins also provide resistance to fusidic acid and crystal violet, but they do so by sequestering these compounds (3,26). CAT acetylates chloramphenicol once or twice; neither the monoacetate nor the diacetate form of chloramphenicol has been shown to have antibiotic activity (27). Most of the studies of chloramphenicol and CAT have been carried out with either gram-negative or gram-positive bacteria. Little is known about the activities of chloramphenicol and CAT in spirochetes, which are distinct from other bacteria in several characteristics (24,32).Spirochaeta aurantia is a pigmented, free-living spirochete found in aquatic environments. In comparison to most other known spirochetes, S. aurantia has simple growth requirements and a fast doubling time (5). These features make it a suitable model organism for genetic studies of spirochetes, and accordingly, we began development of a genetic system for S. aurantia. We had previously shown that a CAT gene of gram-positive bacteria could be expressed in transfected Borrelia burgdorferi (28, 30), and S. aurantia was known to be susceptible to chloramphenicol (4). Thus, there was reason to expect that the CAT gene would provide for positive selection and function as a reporter in S. aurantia as well. However, in preliminary experiments we found that S. aurantia was not only susceptible to chloramphenicol it was also unexpectedly susceptible to diacetyl chloramphenicol, the product of CAT.In the present study we characterized this phenomenon in more detail and investigated the ...

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Chloramphenicol resistance in Streptomyces: cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae

Cited by 38 publications

References 27 publications

Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

Spirochaeta aurantia Has Diacetyl Chloramphenicol Esterase Activity

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