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)

The growing status of E.coli with and without resistance in solid media with different concentration of tetracycline (Tc) is shown above.

Experiment 2(in vivo/qualitative):

After 30mins reaction at 30℃, tetracycline solution discolors when TetX is added which indicates its degradation, while color of the control group remains the same. There were four reaction systems in experimental group which contained respectively 0, 50, 100, 200, 500, 1000uM tetracycline, 2mM NADPH, 100 uM Tris and tetX. Control group contained all the same components of the experimental group but without tetX, and pH of both reaction systems was 8.5. After 30 mins in 30 ℃, the color change of the experimental group(left in figure below) was very obvious, but color of the control group(right in figure below) did not changed obviously.

Experiment 3(in vivo/quantitative):

Enzyme activity test of tetX

100uL reaction system

Tris pH=8.5 10mM

Tc 30µM

TetX 10uL 2.3µM

NADPH 200µM

NADPH is added at last to initiate the reaction

We use the kinetics mode in ultraviolet spectrophotometer to record the changes of absorption in 360nm with total time 200 seconds and cycle time 2 seconds. The addition of NADPH at 25 s increases the absorption at 360nm dramatically. Except for adding no TetX protein, other composition of the control group is the set as the same with experiment group.

The characteristic absorption peak of tetracycline at 360nm in experiment group decreases rapidly, while control remains the same, which indicates the fast reaction of TetX and tetracycline in presence of NADPH. Considering of the ratio of tetracycline and NADPH, 1:1, the interference caused by NADPH absorption was eliminated. Culculation showed the turnover number (Kcat) was 12.6/min.

With the reaction time passes, the characteristic absorption peak of tetracycline at 360nm in experiment group decreases gradually compared with control group.

After 200 seconds, the characteristic absorption peak of tetracycline at 360nm in experiment group drops obviously as compared to 25s when the reaction is initiated by NADPH, while the absorption at 360nm in control group still shows no difference between 25s and 200s.

Kinetics analysis according to Michaelis–Menten Equation

With construct double-reciprocal plot, according to Lineweaver-Burk equation,

mapping 1/Vo to 1/[So], and linear fitting results showed the vertical intercept 1/Vmax and the slope Km/Vmax. Through calculation, Km=48.93uM, Vmax=3.20uM/s; kcat/Km=4.293*103M-1 s-1.

Experiment 4(in vivo/quantitative): E.coli degradated Tc. in M9 medium

100mL M9 liquid medium with 100uM Tc. and 1mM Glucose E.coli expressing tet X and Ruby were respectively added into experimental and control group.(double dilute of the bacterium solution) After 19h or so in shaker under 37℃, tetracycline in the reaction systems was extracted out by HLB solid phase extraction column, and residues of tetracycline in the media could be tested by LC-MS. The chromatograms of control(red) and experiment(green) are as followed: E.coli degraded tetracycline in M9 medium(chromatographic analysis of control and experiment group) By calculated integral area, residue of the experimental group was 1.89% of that of the control group.

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Take the reaction system before experiment as standard system and measure the amount of tetracycline. The amount of Tc.: control/standard system = 60%, experiment/standard = 1.1%

(Note: Compare the peak and calculated integral area at 6.72min(retention time), and the area of other parts could be deleted. Since the liquid system is nine times concentrated, the residue of experimental group was 1.89%)

Different concentration of E.coli solution's tetracycline degradation effect in M9 medium 1)5mL medium with 1mM Glucose

   Concentration gradient of tetracycline: 20uM, 100uM, 200uM

2)5mL medium with 1mM Glucose

   Concentration gradient of tetracycline: 10 times dilution, 20times dilution, 50times dilution

Group 1) and 2) through a night and their changes were as followed:

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