Difference between revisions of "Part:BBa K2150101"

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Tetracycline resistance protein from Bacteroides fragilis

Usage and Biology

Among three dominant tetracycline resistance mechanisms, enzymatic inactivation of tetracycline is a novel type of resistance rather than extensively studied mechanism, efflux and ribosomal protein, which shows great potential in antibiotics degradation. TetX gene is the only thoroughly studied resistance gene initially found in Bacteroides fragilis, coding for a flavin-dependent monooxygenase Tet X that modifies tetracyclines and requires NADPH, Mg2+, and O2 for activity.[1]

Degradation Mechanism

TetX monooxygenase catalyzes regioselective hydroxylation at carbon 11a of tetracyclines. In solutions of pH greater than 1, the product 11a-hydroxytetracycline can decomposes rapidly and non-enzymatically into products that are not easily identifiable. [1]

The monooxygenase reaction mechanism relies on the redox properties of FAD. After reduction to FADH2 by NADPH, the isoalloxazine binds molecular oxygen to form a hydroperoxide. FAD hydroperoxide is formed after substrate recognition, which subsequently direct substrate hydroxylation takes place.[2]

Expression, Purification and SDS-PAGE

We transferred plasmid containing BBa_K2150101 into Escherichia coli BL21 (DE3). The tetracycline resistance protein TetX monooxygenase is expressed under 18℃ in aerobic environment. TetX monooxygenase contains 388 amino acids, the molecular weight of which is 43.7 kDa. The SDS-PAGE of the Ni-NTA His tag purification of the TetX monooxygenase is shown in the figure below.

Activity Analysis of TetX

Experiment 1(in vivo/qualitative):

E. coli having tetX sequence can survive in relatively high tetracycline environment while non-resistant E. coli cannot. Three solid media which contained respectively 0, 2.5, 5 ug/ml tetracycline were inoculated with two kinds of E. coli(one with protein expression of tetX and the other without it), and the concentration of the E. coli solution was in a series of dilution(1x, 10x, 10^2x, 10^3x, 10^4x, 10^5x)

Characterization: In vivo qualitative experiements

References

[1] Ian F. Moore, Donald W. Hughes, and Gerard D. Wright. Tigecycline Is Modified by the Flavin-Dependent Monooxygenase TetX. Biochemistry.44, 11829-11835 (2005)

[2] Gesa Volkers, Gottfried J. Palm, Manfred S. Weiss, Gerard D. Wright, Winfried Hinrichs. Structural basis for a new tetracycline resistance mechanism relying on the TetX monooxygenase. FEBS Letters. 585, 1061-1066(2011)

[3] Brenda S. Speer and Abigail A Salyers. Novel Aerobic Tetracycline Resistance Gene That Chemically Modifies Tetracycline. Journal of Bacteriology. 171.148-153 ( 1989 )

Sequence and Features