6
Bacterial Cellulose
fraction. in. the. presence. of. guanosine. triphosphate. (GTP). stimulated. this.
activity.(Aloni.et.al..1982)..The.protein.that.activated.cellulose.synthase.activity.in.the.presence.of.GTP.was.shown.to.convert.GTP.to.a.cyclic.nucleotide.
that.was.identified.as.cyclic.diguanylate.(c-di-GMP),.the.activator.of.cellulose.
synthase.(Ross et.al..1987)..The.identification.of.c-di-GMP.as.an.activator.of.
cellulose.synthase.led.to.the.synthesis.of.cellulose.in.sufficient.amounts.from.
cell-free. extracts. and. this. allowed. characterization. of. the. enzyme. and. the.
. cellulose.produced.in.vitro..The.presence.of.c-di-GMP.in.the.reaction.mixture.
increased.cellulose.synthase.activity.50–100-fold,.and.using.this.activator.in.
the. reaction. mixture,. it. was. clearly. demonstrated. that. this. activity. resides.
in.the.inner.membrane.in.G. xylinus.(Bureau.and.Brown.1987)..In.addition,.
a.significant.quantity.of.the.cellulose.product.was.synthesized.in.vitro.such.
that. it. could. be. characterized. by. X-ray. diffraction,. and. it. was. shown. to. be.
cellulose II.. A. robust. assay. for. cellulose. synthase. activity. in. vitro. allowed.
further.characterization.of.this.enzyme.and.it.was.shown.that.this.activity.
could.be.solubilized.from.the.membrane.fraction.using.various.detergents..
Solubilization.of.the.cellulose.synthase.activity.followed.by.product.entrapment. aided. further. in. the. purification. of. this. enzyme,. and. in. strain. ATCC.
53582.of.G. xylinus.two.polypeptides.of.molecular.weight.(MW).83.kD.and.
93 kD. were. identified. in. the. purified. fraction. (Lin. and. Brown. 1989).. That.
the.cellulose.synthase.activity.was.associated.with.these.polypeptides.was.
shown.using.5-azido-UDP-glucose.as.a.substrate,.and.it.was.found.that.the.
83.kD.polypeptide.bound.to.the.substrate.and.therefore.was.the.catalytic.subunit.of.cellulose.synthase.(Lin.et.al..1990)..Purification.of.cellulose.synthase.
activity.in.G. xylinus.1306-3.allowed.identification.of.polypeptides.of.MW.90,.
67,.and.54.kD.and.the.67.kD.polypeptide.was.shown.to.bind.c-di-GMP.(Mayer.
et.al..1991)..The.sequence.of.this.polypeptide.was.homologous.to.the.93.kD.
polypeptide.identified.in.G. xylinus.ATCC.53582.and.so.the.93.kD.polypeptide.
was.believed.to.be.the.c-di-GMP.subunit.of.the.cellulose.synthase..However,.
analysis. of. c-di-GMP-binding. proteins. from. a. number. of. bacterial. species.
now.suggests.the.role.of.the.PilZ.domain.in.the.binding.of.c-di-GMP.by.these.
proteins.(Amikam.and.Galperin.2006)..Interestingly,.this.domain.is.present.in.
the.cellulose.synthase.catalytic.subunit.and.not.in.the.93.kD.. protein.that.was.
originally.proposed.to.be.the.c-di-GMP.subunit.
Genes for Cellulose Biosynthesis can be Classified into
Three Groups Based on the Function of the Codified Proteins
Genes. for. cellulose. biosynthesis. can. be. grouped. into. three. main. classes:.
those. that. are. required. for. synthesis. of. the. cellulose. synthase. substrate.
(UDP-glucose),. those. that. are. dedicated. to. cellulose. biosynthesis. only.