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
CARBON SOURCE
PHB
N-ACETYLGLUCOSAMINE
MALTOSE
GLUCONATE
3HB
LACTATE
PYRUVATE
GLUCOSE
B
8.00
Glucose
5.00
7.00
Enzyme
4.00
PHB
6.00
3.00
Protein
5.00
2.00
4.00
1.00
3.00
0.00
2.00
-1.00
1.00
-2.00
0.00
0
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.
FIGURE 1. CONTACT-INDEPENDENT
INDUCTION OF PHB DEPOLYMERASE
A
ACTIVITY
(Units/ml)
8.98
1.83
1.45
0.63
7.51
0.59
1.55
0.00
6.00
24
48
72
96
120
144
CELL PROTEIN x 10 (mg/ml)
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,
3HYDROXYBUTYRATE (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 2. GLUCOSE REPRESSION OF PHB
DEPOLYMERASE SYNTHESIS
GLUCOSE, PHB (g/liter); ACTIVITY
(units/ml)
TABLE 1. EFFECT OF CARBON SOURCE ON
PHB DEPOLYMERASE SYNTHESIS
BACKGROUND
168
TIME (hrs.)
SNC BROTH CONTAINING 0.2% PHB PLUS 0.5%
GLUCOSE WAS INOCULATED (10% v/v INOCULUM)
AND INCUBATED WITH AERATION AT 30C. 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WERE WITHDRAWN AT TIME INTERVALS AND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.
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.
4.0
0 mM 3HB
1.00 mM 3HB
10.0 mM 3HB
0.2% PHB
ENZYME ACTIVITY (Units/ml)
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.
0.10 mM 3HB
5.00 mM 3HB
20.0 mM 3HB
3.0
2.0
1.0
0.0
0
10
20
30
40
50
TIME (hrs)
FIGURE 2. EFFECT OF CELL DENSITY ON PHB
DEPOLYMERASE SYNTHESIS
20
0.07 mg/ml
0.4 mg/ml
ACTIVITY (units/ml)
15
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.
0.7 mg/ml
1.4 mg/ml
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).
2.1 mg/ml
10
CONCLUSIONS
NO PHB
5
FUTURE WORK
0
0
10
20
30
40
50
60
70
80
TIME (hrs.)
CELLS WERE GROWN IN NUTRIENT BROTH AT
30C 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 30C. 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.
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.
REGULATORY SEQUENCES INVOLVED IN REPRESSION AND INDUCTION.
IDENTIFY UPSTREAM
ACKNOWLEDGMENTS
THIS 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.