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Shared strategies for β-lactam catabolism in the soil microbiome.

Nature chemical biology (2018-05-02)
Terence S Crofts, Bin Wang, Aaron Spivak, Tara A Gianoulis, Kevin J Forsberg, Molly K Gibson, Lauren A Johnsky, Stacey M Broomall, C Nicole Rosenzweig, Evan W Skowronski, Henry S Gibbons, Morten O A Sommer, Gautam Dantas
ABSTRACT

The soil microbiome can produce, resist, or degrade antibiotics and even catabolize them. While resistance genes are widely distributed in the soil, there is a dearth of knowledge concerning antibiotic catabolism. Here we describe a pathway for penicillin catabolism in four isolates. Genomic and transcriptomic sequencing revealed β-lactamase, amidase, and phenylacetic acid catabolon upregulation. Knocking out part of the phenylacetic acid catabolon or an apparent penicillin utilization operon (put) resulted in loss of penicillin catabolism in one isolate. A hydrolase from the put operon was found to degrade in vitro benzylpenicilloic acid, the β-lactamase penicillin product. To test the generality of this strategy, an Escherichia coli strain was engineered to co-express a β-lactamase and a penicillin amidase or the put operon, enabling it to grow using penicillin or benzylpenicilloic acid, respectively. Elucidation of additional pathways may allow bioremediation of antibiotic-contaminated soils and discovery of antibiotic-remodeling enzymes with industrial utility.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
6-Nitro-3-(phenylacetamido)benzoic acid, ≥98%
Sigma-Aldrich
Penicillinase from Bacillus cereus, lyophilized powder, 1,500-3,000 units/mg protein (using benzylpenicillin)
Sigma-Aldrich
L-Glutamic acid γ-(4-nitroanilide), γ-glutamyl transpeptidase substrate
Sigma-Aldrich
Penicillin G sodium salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride, ≥98%
Sigma-Aldrich
M9, Minimal Salts, 5X, powder, minimal microbial growth medium
Sigma-Aldrich
Penicillin Amidase from Escherichia coli, 5-10 units/mg protein
Sigma-Aldrich
Phenylacetic acid, ≥99%, FCC, FG
Sigma-Aldrich
(+)-6-Aminopenicillanic acid, 96%
Sigma-Aldrich
4-Nitroaniline, ≥99%
Sigma-Aldrich
4-Nitroacetanilide, 98%