23
Effect of Cultivation on BNC Biomaterials
This. further. supports. the. fact. that. the. formation. of. ketogluconic. acids. is.
not.optimal.for.BNC.production..If.fructose.is.used.as.the.carbon.source.in.
growth.media,.only.cellulose,.acetan,.and.carbon.dioxide . are.produced.(Ross.
et.al..1991)..Studies.have.shown.that.fructose.utilization.is.suppressed.when.
G. xylinus.is.cultivated.in.glucose-based.growth.media,.which.is.not.the.case.
in.sucrose-based.media..This.is.probably.because.glucose.has.an.inhibitory.
effect. on. fructokinase. via. the. competition. with. fructose.. Glucose. is. therefore. physiologically. significant,. as. it. may. serve. as. a. sensitive. regulatory.
. mechanism. (Benzamin. and. Rivertz. 1972).. On. the. other. hand,. glucose. can.
be.utilized.as.a.substrate.even.if.fructose.or.sucrose.is.added.to.the.growth.
media.as.the.main.carbon.source.(Benzamin.and.Rivertz.1972).
The.initial.metabolic.steps.in.the.fructose.and.glucose.utilization.pathways.
are. initially. different,. whereas,. from. glucose-6-phosphate. and. further,. the.
same. metabolic. pathway. is. followed. for. BNC. production.. Glucose. is. taken.
up.by.the.bacteria.and.transformed.into.glucose-6-phosphate.by.gluco. kinase..
Fructose,. on. the. other. hand,. is. first. phosphorylated. into. fructose-6. phosphate.by.fructokinase.and.thereafter.transformed.into.glucose-6-phosphate.
by. phosphoglucoisomerase.. Glucose-6-phosphate. is. transformed. via. isomerization. into. glucose-1-phosphate. by. phosphoglucomutase. followed. by.
transformation. of. glucose-1-phosphate. into. uridine. diphosphate. glucose.
(UDP-glucose). by. pyrophosphorylase. UDPGlc. (Ross. et. al.. 1991).. G. xylinus.
mutants. that. are. cellulose. negative. (Cel − ). lack. pyrophosphorylase. UDPGlc.
and.can.therefore.not.produce.UDP-glucose.and.BNC,.since.UDP-glucose.is.
a.crucial.inter. mediate.for.cellulose.synthesis.(Krystynowicz.et.al..2005)..The.
organization. of. . cellulose. synthase. in. the. membrane. is. important. for. regulating. diverse. cellular. functions,. including. motility,. biofilm. formation,. cell.
cycle.progression,.and.virulence.in.bacteria..Cellulose.synthase.is.regulated.
by. the. cyclic. nucleotide. bis-(3′,5′)-cyclic. dimeric. guanosine. monophosphate.
(c-di-GMP),.which.acts.as.a.reversible.allosteric.activator.for.the.enzyme;.that.
is,.when.c-di-GMP.binds.cellulose.synthase.(at.a.site.distant.to.the.active.site),.
it.enhances.the.activity.of.the.enzyme..The.concentration.of.c-di-GMP.is.in.
phodiesterase.A.and.B.(PDE-A.and.PDE-B).that.degrade.c-di-GMP..The.degradation.mechanism.of.c-di-GMP.by.PDE-A.and.PDE-B.is.still.unclear.(Ross.
et.al..1990)..PDE-A.is.Ca 2+ .sensitive.and.studies.have.concluded.that.PDE-A.
is.inhibited.by.Ca 2+ .since.Ca 2+ .is.suggested.to.compete.for.a.binding.site.with.
Mg 2+ ,.which.is.required.for.the.PDE-A.activity.(Ross.et.al..1990)..More.recent.
studies.have.also.demonstrated.that.PDE-A.is.negatively.regulated.by.. oxygen.
due.to.a.heme-containing.domain.that.binds.oxygen.reversibly.(Chang.et.al..
2001)..The.regulating.process.is.not.fully.understood.and.investigations.are.
ongoing.to.unravel.the.full.regulating.process.of.cellulose.synthase.
Many.other.enzymes.and.regulatory.mechanisms.are.involved.in.the.. carbon.
metabolism. of. G. xylinus. and. only. a. fraction. of. them. have. been. mentioned.
here.since.the.focus.of.this.review.is.BNC.production.and.not.the.metabolism.
of.bacteria.in.general.
Effect of Cultivation on BNC Biomaterials
This. further. supports. the. fact. that. the. formation. of. ketogluconic. acids. is.
not.optimal.for.BNC.production..If.fructose.is.used.as.the.carbon.source.in.
growth.media,.only.cellulose,.acetan,.and.carbon.dioxide . are.produced.(Ross.
et.al..1991)..Studies.have.shown.that.fructose.utilization.is.suppressed.when.
G. xylinus.is.cultivated.in.glucose-based.growth.media,.which.is.not.the.case.
in.sucrose-based.media..This.is.probably.because.glucose.has.an.inhibitory.
effect. on. fructokinase. via. the. competition. with. fructose.. Glucose. is. therefore. physiologically. significant,. as. it. may. serve. as. a. sensitive. regulatory.
. mechanism. (Benzamin. and. Rivertz. 1972).. On. the. other. hand,. glucose. can.
be.utilized.as.a.substrate.even.if.fructose.or.sucrose.is.added.to.the.growth.
media.as.the.main.carbon.source.(Benzamin.and.Rivertz.1972).
The.initial.metabolic.steps.in.the.fructose.and.glucose.utilization.pathways.
are. initially. different,. whereas,. from. glucose-6-phosphate. and. further,. the.
same. metabolic. pathway. is. followed. for. BNC. production.. Glucose. is. taken.
up.by.the.bacteria.and.transformed.into.glucose-6-phosphate.by.gluco. kinase..
Fructose,. on. the. other. hand,. is. first. phosphorylated. into. fructose-6. phosphate.by.fructokinase.and.thereafter.transformed.into.glucose-6-phosphate.
by. phosphoglucoisomerase.. Glucose-6-phosphate. is. transformed. via. isomerization. into. glucose-1-phosphate. by. phosphoglucomutase. followed. by.
transformation. of. glucose-1-phosphate. into. uridine. diphosphate. glucose.
(UDP-glucose). by. pyrophosphorylase. UDPGlc. (Ross. et. al.. 1991).. G. xylinus.
mutants. that. are. cellulose. negative. (Cel − ). lack. pyrophosphorylase. UDPGlc.
and.can.therefore.not.produce.UDP-glucose.and.BNC,.since.UDP-glucose.is.
a.crucial.inter. mediate.for.cellulose.synthesis.(Krystynowicz.et.al..2005)..The.
organization. of. . cellulose. synthase. in. the. membrane. is. important. for. regulating. diverse. cellular. functions,. including. motility,. biofilm. formation,. cell.
cycle.progression,.and.virulence.in.bacteria..Cellulose.synthase.is.regulated.
by. the. cyclic. nucleotide. bis-(3′,5′)-cyclic. dimeric. guanosine. monophosphate.
(c-di-GMP),.which.acts.as.a.reversible.allosteric.activator.for.the.enzyme;.that.
is,.when.c-di-GMP.binds.cellulose.synthase.(at.a.site.distant.to.the.active.site),.
it.enhances.the.activity.of.the.enzyme..The.concentration.of.c-di-GMP.is.in.
phodiesterase.A.and.B.(PDE-A.and.PDE-B).that.degrade.c-di-GMP..The.degradation.mechanism.of.c-di-GMP.by.PDE-A.and.PDE-B.is.still.unclear.(Ross.
et.al..1990)..PDE-A.is.Ca 2+ .sensitive.and.studies.have.concluded.that.PDE-A.
is.inhibited.by.Ca 2+ .since.Ca 2+ .is.suggested.to.compete.for.a.binding.site.with.
Mg 2+ ,.which.is.required.for.the.PDE-A.activity.(Ross.et.al..1990)..More.recent.
studies.have.also.demonstrated.that.PDE-A.is.negatively.regulated.by.. oxygen.
due.to.a.heme-containing.domain.that.binds.oxygen.reversibly.(Chang.et.al..
2001)..The.regulating.process.is.not.fully.understood.and.investigations.are.
ongoing.to.unravel.the.full.regulating.process.of.cellulose.synthase.
Many.other.enzymes.and.regulatory.mechanisms.are.involved.in.the.. carbon.
metabolism. of. G. xylinus. and. only. a. fraction. of. them. have. been. mentioned.
here.since.the.focus.of.this.review.is.BNC.production.and.not.the.metabolism.
of.bacteria.in.general.