7
Biosynthesis of Bacterial Cellulose
(cellulose.synthase.and.associated.proteins),.and.those.that.are.required.for.
turnover.(synthesis.and.cleavage).of.the.cellulose.synthase.activator.c-di-GMP..
By.chance,.identification.of.these.three.groups.of.genes.took.place.in.the.same.
order.as.listed.here..Genes.in.the.first.group.were.identified.by.complementation.of.mutants.that.produced.no.pellicle.in.liquid.culture..These.mutants.
produced.smooth.colonies.on.agar.plates.and.were.considered.to.be.cellulose.
negative.(Cel − )..Using.a.genomic.library.of.G. xylinus.for.complementation.of.
these.mutants,.genes.for.UDP-glucose.pyrophosphorylase.(Valla.et.al..1989).
and.phosphoglucomutase.(Fjærvik.et.al..1991).were.identified.and.shown.to.
be.involved.in.cellulose.biosynthesis.because.of.their.role.in.the.synthesis.of.
UDP-glucose.. A. more. detailed. analysis. of. Cel − . mutants. in. G. xylinus. strain.
1306-3.and.their.complementation.using.a.genomic.library.allowed.isolation.
of.a.large.DNA.fragment.that.was.shown.to.carry.four.genes.that.were.organized. as. an. operon,. and. these. genes. were. suggested. to. code. for. the. cellulose.synthase.and.other.proteins.required.for.cellulose.synthesis.in.bacteria.
(Wong. et. al.. 1990).. This. operon. was. called. the. bacterial. . cellulose. synthase.
(bcs).operon,.and.the.genes.were.labeled.as.bcsA,.bcsB,.bcsC,.and.bcsD,.with.
bcsB. suggested. as. the. gene. coding. for. cellulose. synthase.. Direct. identification. and. isolation. of. the. gene. for. cellulose. synthase. from. G. xylinus. strain.
ATCC.53582.was.accomplished.at.the.same.time.using.oligonucleotide.probes.
designed.from.the.N-terminal.amino.acid.sequence.of.the.83.kD.polypeptide.
identified.in.the.purified.cellulose.synthase.(Saxena.et.al..1990)..More.importantly,.the.amino.acid.sequence.derived.from.the.83 kD.polypeptide.coding.
gene.was.found.to.be.homologous.to.the.amino.acid.sequence.derived.from.
the.bcsA.gene.in.the.bcs.operon.and.correctly.identified.it.to.be.the.gene.for.
cellulose.synthase..Sequencing.of.a.region.adjacent.to.the.83.kD.polypeptide.
coding. gene. led. to. the. identification. of. other. genes,. including. that. coding.
for. the. 93. kD. polypeptide. that. was. also. identified. in. the. purified. cellulose.
synthase.(Saxena.et.al..1991)..Although.the.organization.of.genes.for.cellulose.
synthesis.in.G. xylinus.strains.ATCC.53582.and.ATCC.23769.was.similar.to.
what.was.identified.in.the.bcs.operon.in.strain.1306-3,.the.bcsA.and.bcsB.genes.
in.these.strains.were.found.to.be.fused.as.a.single.gene.(acsAB)..Since.this.
organization.of.genes.for.cellulose.synthesis.was.first.identified.in.G. xylinus.
(Acetobacter xylinum.at.that.time),.we.named.the.operon.the.acetobacter.cellulose-synthesizing.operon.(the.acs.operon).in.which.the.genes.were.shown.to.
be.organized.as.acsAB,.acsC,.and.acsD.(Saxena.et.al..1994)..Biochemical.analysis.revealed.that.no.polypeptide.of.the.size.coded.for.by.the.acsAB.gene.could.
be.visualized.in.extracts.of.G. xylinus.ATCC.53582;.instead,.two.polypeptides.
of.83.kD.and.93.kD.were.present.in.the.purified.cellulose.synthase,.suggesting.that.posttranslational.processing.of.a.large.polypeptide.results.in.these.
two.polypeptides..Additionally.these.two.polypeptides.were.present.in.a.1:1.
ratio.in.the.purified.cellulose.synthase.(Chen.and.Brown.1996)..Interestingly,.
a.second.fused.cellulose.synthase.gene.(acsAII).was.identified.in.G. xylinus.
ATCC.23769,.and.although.this.gene.was.not.required.for.cellulose.synthesis,.
cellulose.synthase.activity.determined.by.this.gene.was.observed.in.mutants.