FACTORS INFLUENCING THE SYNTHESIS OF POLYHYDROXYBUTYRATE DEPOLYMERASE IN STREPTOMYCES SP. 5A

Matthew B. Persinger, Matthew Shull, and Stephen F. Baron, Biology Dept., Bridgewater College, Bridgewater, VA 22812

ACKNOWLEDGMENTSTHIS RESEARCH WAS SUPPORTED BY GRANT #J-713 FROM THE THOMAS F. AND KATE MILLER JEFFRESS MEMORIAL TRUST. WE THANK JON KASTENDIEK AND THE JAMES MADISON UNIVERSITY BIOLOGY DEPARTMENT FOR PRINTING THE POSTER.

BACKGROUND

POLYHYDROXYBUTYRATE (PHB) IS ONE OF A GROUP OF POLYMERS CALLED POLYHYDROXYALKANOATES (PHAs), WHICH ARE PRODUCED BY VARIOUS SOIL BACTERIA. THEY CAN BE USED AS PLASTIC SUBSTITUTES, BUT ARE FULLY DEGRADABLE BY SOIL AND WATER BACTERIA. THE ACTINOMYCETE, STREPTOMYCES SP. 5A, SECRETES A PHB DEPOLYMERASE DURING GROWTH ON PHB. THIS ENZYME DEGRADES PHB TO ITS MONOMERIC FORM, 3-HYDROXYBUTYRATE (3HB), WHICH IS THEN METABOLIZED. THE HYDROLYSIS OF PHB PRODUCES CLEARING ZONES IN AGAR MEDIA (FIG. 1). OUR RESEARCH GOAL WAS TO IDENTIFY FACTORS THAT REGULATE SYNTHESIS OF THIS ENZYME.

FIGURE 1. CONTACT-INDEPENDENT INDUCTION OF PHB DEPOLYMERASE

CELLS WERE GROWN ON A MINERAL SALTS AGAR MEDIUM (SNC) CONTAINING 0.2% PHB GRANULES AS THE SOLE CARBON SOURCE. CELLS WERE INOCULATED DIRECTLY ONTO THE SURFACE OF THE AGAR (PLATE A) OR ONTO A THICK OVERLAY OF 3% AGAR IN SNC (PLATE B). FORMATION OF CLEARING ZONES ON BOTH PLATES SUGGESTS THAT DIRECT CONTACT OF THE BACTERIA WITH PHB GRANULES IS NOT NECESSARY FOR ENZYME INDUCTION. THUS, A DIFFUSIBLE INDUCER MUST BE INVOLVED.

A B

TABLE 1. EFFECT OF CARBON SOURCE ON PHB DEPOLYMERASE SYNTHESIS

CELLS (0.7 mg DRY CELL WT./ml) WERE CULTURED FOR 48 HRS. IN SNC BROTH WITH THE ABOVE CARBON SOURCES AT 20 mM (PHB at 0.2% w/v). THE HIGHEST ACTIVITIES WERE OBTAINED WITH PHB AND 3HB. SIGNIFICANT ACTIVITIES WERE OBTAINED WITH N-ACETYLGLUCOSAMINE, MALTOSE, AND CERTAIN ORGANIC ACIDS. GLUCOSE DID NOT SUPPORT ENZYME SYNTHESIS, SUGGESTING THAT CATABOLITE REPRESSION MIGHT BE INVOLVED.

CARBON SOURCE ACTIVITY(Units/ml)

PHB 8.98

N-ACETYLGLUCOSAMINE 1.83

MALTOSE 1.45

GLUCONATE 0.63

3HB 7.51

LACTATE 0.59

PYRUVATE 1.55

GLUCOSE 0.00-2.00

-1.00

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 24 48 72 96 120 144 168

TIME (hrs.)

GLU

CO

SE, P

HB

(g/li

ter)

; AC

TIVI

TY

(uni

ts/m

l)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

CEL

L PR

OTE

IN x

10

(mg/

ml)

Glucose

Enzyme

PHB

Protein

FIGURE 2. GLUCOSE REPRESSION OF PHB DEPOLYMERASE SYNTHESIS

0.0

1.0

2.0

3.0

4.0

0 10 20 30 40 50

TIME (hrs)

EN

ZY

ME

AC

TIV

ITY

(U

nit

s/m

l)

0 mM 3HB 0.10 mM 3HB1.00 mM 3HB 5.00 mM 3HB10.0 mM 3HB 20.0 mM 3HB0.2% PHB

FIGURE 3. EFFECT OF 3HB CONCENTRATION ON PHB DEPOLYMERASE SYNTHESIS

CELLS WERE GROWN IN SNC BROTH CONTAINING THE INDICATED CONCENTRATIONS OF 3HB OR PHB. EVEN AT CONCENTRATIONS AS LOW AS 0.1 mM, 3HB WAS AN EFFECTIVE INDUCER OF ENZYME ACTIVITY. THE LAG TIME FOR INDUCTION WITH 3HB WAS ABOUT 24 HRS. LESS THAN WITH PHB. WE HYPOTHESIZE THAT DURING GROWTH ON PHB, TRACE AMOUNTS OF 3HB ARE GENERATED BY A LOW LEVEL OF CONSTITUTIVELY PRODUCED PHB DEPOLYMERASE. THE 3HB THUS PRODUCED MAY THEN INDUCE FULL SYNTHESIS OF THE ENZYME.

FIGURE 2. EFFECT OF CELL DENSITY ON PHB DEPOLYMERASE SYNTHESIS

CELLS WERE GROWN IN NUTRIENT BROTH AT 30C WITH AERATION OVERNIGHT, WASHED ONCE WITH SNC LIQUID MEDIUM, AND RESUSPENDED IN SNC PLUS 0.2% PHB AT THE CELL DENSITIES INDICATED (mg DRY CELL WT./ml). CULTURES WERE INCUBATED WITH AERATION AT 30C. SAMPLES WERE REMOVED AT TIME INTERVALS, CLARIFIED BY CENTRIFUGATION, AND ASSAYED FOR PHB DEPOLMERASE ACTIVITY BY A TURBIDOMETRIC ASSAY. MAXIMAL ACTIVITY WAS OBTAINED AFTER 48 HRS. WITH AN INITIAL CELL DENSITY OF 0.7 mg DRY CELL WT./ml. THESE CONDITIONS WERE USED FOR MOST OF THE ENZYME INDUCTION EXPERIMENTS DESCRIBED HERE.

0

5

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15

20

0 10 20 30 40 50 60 70 80

TIME (hrs.)

AC

TIV

ITY

(un

its/m

l)

0.07 mg/ml

0.4 mg/ml

0.7 mg/ml

1.4 mg/ml

2.1 mg/ml

NO PHB

CONCLUSIONS

1. CONTACT OF CELLS WITH PHB IS NOT NECESSARY TO INDUCE PHB DEPOLYMERASE SYNTHESIS. A DIFFUSIBLE INDUCER MAY BE INVOLVED.

2. PHB AND 3HB ARE EFFECTIVE INDUCERS OF ENZYME ACTIVITY. ENZYME SYNTHESIS DURING GROWTH ON PHB MAY BE INDUCED BY SMALL AMOUNTS OF 3HB GENERATED BY A LOW LEVEL OF CONSTITUTIVELY PRODUCED PHB DEPOLYMERASE.

3. GLUCOSE REPRESSES PHB DEPOLYMERASE SYNTHESIS, PRESUMABLY BY A CATABOLITE REPRESSION MECHANISM. GLUCOSE REPRESSION IN STREPTOMYCES IS THOUGHT TO INVOLVE A REGULATORY GLUCOSE KINASE (KWAKMAN AND POSTMA, 1994, J. BACTERIOL., 176:2694-2698).

SNC BROTH CONTAINING 0.2% PHB PLUS 0.5% GLUCOSE WAS INOCULATED (10% v/v INOCULUM) AND INCUBATED WITH AERATION AT 30C. SAMPLES WERE WITHDRAWN AT TIME INTERVALS AND ASSAYED FOR ENZYME ACTIVITY, PHB CONTENT, GLUCOSE, AND CELL PROTEIN. PHB DEGRADATION AND ENZYME SYNTHESIS BEGAN ONLY WHEN GLUCOSE WAS COMPLETELY METABOLIZED, INDICATING CATABOLITE REPRESSION.

FUTURE WORK

1. ISOLATE GLUCOSE KINASE MUTANTS OF STREPTOMYCES SP. 5A AND DETERMINE WHETHER THEY ARE DEFICIENT IN GLUCOSE REPRESSION OF PHB DEPOLYMERASE SYNTHESIS.

2. DO FURTHER EXPERIMENTS TO CONFIRM THAT 3HB IS THE DIFFUSIBLE INDUCER.

3. CLONE AND SEQUENCE THE GENE ENCODING PHB DEPOLYMERASE. IDENTIFY UPSTREAM REGULATORY SEQUENCES INVOLVED IN REPRESSION AND INDUCTION.