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MINISTERIO DE COMERCIO EXTERIOR PUBLICACIÓN DE PRIMERA VEZ

RÉGIMEN MUNICIPAL

MINISTERIO DE COMERCIO EXTERIOR PUBLICACIÓN DE PRIMERA VEZ

Although.there.may.be.general.agreement.on.the.geochemical.events.on.early.Earth,.some.of.which.

are.based.on.laboratory.experiments.that.attempted.to.reproduce.conditions.of.substrate.chemicals,.

temperature,.pressure,.electrical.discharge,.etc.,.there.is.general.disagreement.on.what.caused.life.

to.appear..Some.scientists.believe.that.life.arose.spontaneously.about.4.billion.years.ago.and.has.

evolved.to.what.we.see.today..Other.persons.of.faith.believe.that.God.created.life,.about.4.billion.

years.ago,.and.through.the.mechanisms.of.evolution,.which.he.(or.she).also.created,.life.has.evolved.

throughout.the.millennia.to.our.present.biosphere..These.beliefs,.of.course,.contradict.the.biblical.

account.given.in.Genesis.I.in.the.Bible.and.contradict.the.position.of.“Young.Earthers,”.who,.on.

faith,.believe.that.all.life-forms.(including.humans).were.created.in.one.short.period.on.Earth,.some.

6,000.years.ago.

In.the.following.text,.we.will.discuss.two.current,.plausible,.nonfaith.theories.for.the.appear-ance.of.the.first.life.on.Earth..These.are.the.theories.of Directed Panspermia.(DP).and.Abiogenesis (or Biopoesis).

The.theory.of.DP.presented.by.Crick.[1981].and.Orgel.[1973].argues.that.it.would.have.been.

almost.impossible.to.start.life.on.Earth,.given.the.biochemical.complexity.of.even.the.simplest.pro-karyotes.and.Archaea.today..Sadly,.DP.puts.off.the.problem.of.understanding.and.describing.how.

life.did.originate.and.evolve.on.Earth..DP.argues.that.life.evolved.or.was.created.elsewhere.in.the.

universe.and.came.to.Earth.either.by.cosmic.dust,.meteorites.(on.their.interior),.or.by.a.“spaceship.”.

This.life.must.have.been.very.primitive.and.very.rugged.to.withstand.a.journey.in.the.vacuum.and.

cold.of.space,.and.the.heat.of.reentry.if.the.life-form.was.in.a.meteorite.

One.argument.for.DP.is.that.all.life.today.(Prokaryotes,.Archaeans,.and.Eukaryotes).uses.a.

nearly.universal.dsDNA.genetic.code.read.out.by.RNA..That.there.is.little.variation.in.this.basic.

design.is.said.to.argue.against.development.of.life.on.Earth,.which.supposedly.would.have.led.to.

more.variability.in.the.genomic.code.for.proteins..Or,.was.the.initial.design.so.robust.that.no.other.

genomic.design.could.effectively.compete?.In.any.case,.if.one.subscribes.to.DP,.one.still.has.to.

explain.how.life.arose.elsewhere.in.the.universe.

Abiogenesis. theorizes. that. primitive. life. was. self-started. on. early. Earth. by. really. fortuitous.

chemical.reactions..Conditions.on.the.recently.cooled.planet.first.gave.rise.to.organic.monomers..

Then.over.the.millennia,.biological.polymers.arose,.including.RNA.and.structural.phospholipids..

Here.is.the.big.jump;.the.organic.substrates.developed.self-replication.and,.finally,.some.sort.of.

protective.cell.membrane.

In.the.1970s,.Manfred.Eigen.at.the.Max.Planck.Institute.examined.the.transient.stages.between.

the.molecular.chaos.in.the.prebiotic.“soup”.and.the.transient.stages.of.a.self-replicating.hypercycle between.the.molecular.chaos.of.the.prebiotic.soup.and.simple,.macromolecular,.self-replicating.sys-tems..It.was.postulated.that.in.a.hypercycle,.the.information.storage.system.(ISS).(probably.RNA).

produced.an.enzyme.that.catalyzed.the.formation.of.another.information.system,.in.sequence,.until.

the.product.of.the.last.ISS.aided.in.the.formation.of.an.ISS,.similar.to.the.first.system..Perhaps,.

ribozyme-like.enzymes.mediated.the.catalysis.required.in.the.hypercycle..Still.missing.is.the.devel-opment.of.the.enclosure.of.the.hypercycle.system.by.a.membrane.and.the.coding.of.proteins.by.

nucleic.acids.

0 Introduction to Molecular Biology, Genomics and Proteomic for Biomedical Engineers One.slant.on.abiogenesis.concerns.the RNA World..This.term.was.coined.in.1986.by.Walter.

Gilbert.writing.on.the.then-recent.observations.about.the.catalytic.properties.of.RNA.ribozymes [Kimball.2005q]. The.RNA.World.hypothetically.took.place.in.a.span.of.about.500.million.years,.

from.3.5.billion.to.3.0.billion.years.ago..During.this.period,.proteins.and.protein.enzymes.did.not.

yet.figure.in.biochemical.reactions,.and.RNA.was.postulated.to.carry.both.the.genetic.information.

storage.and.perform.the.catalysis.necessary.in.primitive,.self-replicating,.organic.chemical.systems..

Gilbert.noted.that.neither.DNA.nor.proteins.were.required.in.such.a.primitive.organic.chemical.

system..A.major.difficulty.in.accepting.the.RNA.World.scenario.is.modeling.the.transition.from.

RNA-based.information.storage.and.catalysis.to.DNA.information.storage.and.protein.catalysis..

One.such.RNA → DNA + Protein.molecular.scenario.has.been.proposed.by.Line.[2005].

We.note.that.DNA.is.much.more.suitable.for.genomic.information.storage;.it.is.chemically.more.

stable.than.RNA..So,.did.some.sort.of.early.reverse transcriptase.copy.original.RNA.information.to.

DNA.molecules?.Modern.rRNA.is.found.in.association.with.many.proteins,.but.it.is.the.rRNA.that.

catalyzes.the.assembly.of.polypeptides,.not.the.proteins.

We.have.several.difficulties.in.accepting.the.validity.of.the.RNA.World.hypothesis..Neither.

nucleotides.nor.ribose.were.found.in.the.Urey–Miller.reactor.experiments..Somehow,.they.would.

have.to.be.assembled.from.their.components:.deoxyribose;.the.bases.A,.T,.G,.and.C;.and.phos- phates..This.assembly.involves.the.enzyme-directed.expenditure.of.chemical.energy.to.form.cova-lent bonds between.the.bases. Only.8%.of.purines.will.bind.to.the.1′.carbon.of.ribose.spontaneously,.

under.anhydrous.conditions..Pyrimidine.bases.will.not.spontaneously.bind.to.the.1′.carbon.of.ribose.

anhydrously..Bases.do.not.react.spontaneously.with.ribose.in.aqueous.media..The.base.cytosine.

easily.undergoes.hydrolysis.and.has.no.plausible.prebiotic.synthesis.scenario..Phosphate.ions.are.

rare.because.they.precipitate.with.calcium.and.carbonate.ions..Thus,.it.appears.to.us.that.the.RNA.

World.hypothesis.is.built.on.shaky.ground.

The.Oparin.theory.of.coacervates.describes.how.at.the.correct.pH.and.ionic.milieu,.colloidal.

organic.particles.form.a.closed.surface.(the.process.of.coacervation).around.a.central.volume..This.

coacervation.formed.a.protective,.protocell.membrane..By.isolating.the.inner.volume,.the.chemical.

reactions.therein.were.protected.and.could.have.better.access.to.the.molecular.substrates.needed,.

which.penetrated.the.membrane..There.lies.the.rub;.how.did.these.primitive.boundaries.keep.out.

the.“bad”.molecules.and.let.in.the.“good”.ones?.Such.mechanisms.are.seen.in.modern,.eukaryotic.

cells..Complex.protein.receptors.and.“gatekeepers”.are.embedded.in.the.cell.membranes.to.regulate.

ionic.and.molecular.in/out.traffic..Note.that.there.is.a.quantum.leap.in.complexity.between.a.coac-ervate.“cell”.and.the.actual,.sophisticated.cell.membrane.structures.seen.in.contemporary.archaean,.

prokaryote,.and.eukaryote.cells.

Another.puzzler.in.contemplating.the.“spontaneous”.development.of.cells.is.the.nucleic.acid–

enzyme.problem..Perhaps,.earliest.life.used.available.metal.carbides.and.oxides.as.catalysts.of.their.

chemical.reactions..In.a.modern.cell,.the.genomic.readout.and.cell.reproduction.are.promoted.by.

protein.and.RNA.enzymes.whose.structures.are.very.specific,.and.in.turn,.are.coded.in.the.DNA.

genome.of.the.cell..Did.primitive,.short.DNA.molecules.code.randomly.at.first.until.a.protein.struc-ture.was.found.that.increased.the.survival.probability.of.a.particular.DNA.gene?.And.what.about.

the.role.of.RNA.in.early.protocells?.Which.came.first,.the.mRNA.or.the.DNA?.Life.today.generally.

uses.d-isomer.sugars.(relative.to.d-glyceraldehyde).and.builds.proteins.out.of.l-amino.acids..(One.

exception.is.seen.in.the.molecular.chirality preferences.of.contemporary.Archaeans.)

Kimball. [2004a]. points. out. that. the. (present). synthesis. of. DNA. and. RNA. requires. protein.

enzymes;. nucleic. acids. cannot. be. made. without. proteins,. and. proteins. cannot. be. made. without.

nucleic.acids.(the.classic.chicken or egg first paradox)..Kimball.writes.that.we.now.know.that.cer-tain.RNA.molecules.called.ribozymes.incorporate.two.major.features.necessary.for.life:.storage.of.

information.(in.their.Nt.base.sequence).and.the.ability.to.act.as.catalysts.

Kimball.states:.“While.no.ribozyme.in.nature.has.yet.been.found.that.can.replicate.itself,.ribo- zymes.have.been.synthesized.in.the.laboratory.that.can.catalyze.the.assembly.of.short.oligonucle-otides.into.exact.complements.of.themselves..The.ribozyme.serves.as.both.the.template.on.which.

short. lengths. of. RNA. ‘oligonucleotides’. are. assembled. following. the. rules. of. base. pairing. and.

the.catalyst.for.covalently.linking.these.oligonucleotides..In.principal,.the.minimal.functions.of.

life.might.have.begun.with.RNA.and.only.later.did.proteins.take.over.the.catalytic.machinery.of.

metabolism.and.DNA.takes.over.as.the.repository.of.the.genetic.code..Several.other.bits.of.evidence.

support.this.notion.of.an.original.‘RNA.world’:.Many.of.the.cofactors.that.play.so.many.roles.in.

[contemporary].life.are.based.on.the.[sugar].ribose;.for.example:.ATP,.NAD,.FAD,.coenzyme.A,.

cyclic.AMP,.GTP..In.the.cell,.all.deoxyribonucleotides.are.synthesized.from.ribonucleotide.precur-sors..Many.[contemporary].bacteria.control.the.transcription.and/or.translation.of.certain.[DNA].

genes.with.RNA.molecules,.not.protein.molecules.”.[This,.and.all.other.quotes.from.J..Kimball’s.

Biology Pages®,.are.used.with.his.kind.permission.]

An.interesting.speculation,.but.why.does.all.contemporary.life.now.use.dsDNA.for.information.

storage.and.relegate.RNA.to.executive.roles?

Clearly,. the. study. of. biochemical. evolution. is. highly. speculative.. There. is. a. fossil. record. of.

early.prokaryotes.found.in.sandstone.dating.about.3.8.billion.years.ago..Unfortunately,.fossils.tell.

us.nothing.about.a.cell’s.molecular.biology..We.only.have.evidence.from.comparative.cell.biochem-istry,.the.genome.structures,.and.the.proteins.of.various.contemporary.life-forms..This.evidence.is.

used.to.make.informed.conjectures.as.to.what.life-form.evolved.to.what.over.the.millennia.to.create.

evolutionary.“trees.”

Some.of.the.earliest.traces.of.early.prokaryotic.life.are.found.fossilized.in.rocks.isotopically.

dated.at.about.3.8.billion.(3.8.×.109 ).years.of.age..These.fossilized.cells.resemble.present-day.cya-nobacteria.. Modern. cyanobacteria. are. prokaryotes. that. have. an. elaborate. and. highly. organized.

system.of.internal.membranes.which.function.in.photosynthesis..Blue-green.cyanobacteria.capture.

photons.and.use.them.to.drive.their.metabolic.engines..Chlorophyll.IIA.and.some.accessory.pigment.

molecules.(phycocyanin.and.phycoerythrin).are.embedded.in.their.photosynthetic.lamellae..These.

pigments.give.the.cyanobacteria.a.wide.spectrum.of.colors:.red,.yellow,.green,.blue-green,.deep.

blue,.and.violet.cyanobacteria.are.known..Cyanobacteria.can.be.single.celled.or.colonial..Depend-ing. on. the. environment. and. the. species,. they. can. form. filaments,. sheets,. or. hollow. balls.. They.

may. even. form. spores. under. harsh. environmental. conditions.. Cyanobacteria. also. form. complex.

symbiotic.relationships.with.many.fungi,.forming.lichens..Other.cyanobacterial.symbioses.include.

certain.protists,.marine.sponges,.diatoms,.echiuroid.worms,.angiosperms,.cycads,.pteridophytes,.

liverworts,.and.mosses..Cyanobacteria.reproduce.by.binary.fission,.budding,.or.fragmentation.

Lindell.et.al..[2004,.2005].have.reported.that.the.marine.phage,.Podovirus.P-SSP7,.infects.the.

marine. cyanobacterium,. Prochlorococcus. This. in. itself. is. not. remarkable,. but. the. fact. that. the.

phage.virus.carries.photosynthesis.genes.in.addition.to.the.genes.it.needs.to.reproduce.itself.in.a.

cyanobacterial.host.is..Of.course,.the.phage.virion.does.not.practice.photosynthesis.or.any.other.

type.of.metabolism;.for.that.matter,.it.is.not.alive..The.phage’s.photosynthesis.genes.have.overlap-ping.start.and.stop.codons.and.are.transcribed.along.with.essential.phage.capsid.genes,.suggesting.

that.they.are.integral.parts.of.the.viral.DNA.genome..Both.the.phage.and.the.cyanobacterium.carry.

the.hli high.light-inducible.genes.for.proteins.that.protect.the.bacterium’s.photosynthetic.machinery.

from.light.damage..They.both.also.carry.psbA,.a.gene encoding.the.photosystem PSII.core.reaction.

protein.D1. Cyanobacteria.photosynthesis.uses.the.Photosynthesis II.(PS-II).process..Experimental.

evidence.indicates.that.the.viral.genome.boosts.photosynthesis.in.the.host.cell,.giving.the.cyano-bacteria.more.energy.(ATP).and.thus.a.longer.time.for.the.phage.to.reproduce.inside.it,.ultimately.

leading.to.more.phages.on.viral.eclosion..One.wonders.how.many.other.viruses.bring.“gifts”.to.their.

hosts.to.enhance.host.stamina.in.support.of.viral.reproduction.

Probably.the.most.important.contribution.to.life.on.Earth.by.this.3.5-billion-year-old.class.of.

bacteria.is.its.ability.to.produce.molecular oxygen.(O2 ).by.photosynthesis..Thanks.to.this.photo-synthesis.in.the.Archaean.and.Proterozoic eras,.the.O2.concentration.in.Earth’s.atmosphere.began.

to.rise.about.2.2.billion.years.ago..It.rose.slowly.because.the.free.O2.formed.oxides.with.exposed,.

reactive.minerals.in.Earth’s.crust..Iron.oxides.were.formed,.which.show.up.today.as.red.layers.in.

sandstone.about.2.billion.years.old..This.mineral.buffering.of.O2.went.on.for.perhaps.1.4.billion.

 Introduction to Molecular Biology, Genomics and Proteomic for Biomedical Engineers years.until.there.was.little.exposed.elemental.iron.left.to.oxidize..At.that.point,.about.800.million.

years.ago,.the.atmospheric.O2.partial.pressure.rose.abruptly.to.about.20%..It.spiked.again.to.a.peak.

of.about.37%.about.400.million.years.ago,.no.doubt.owing.to.the.flourishing.of.photosynthetic.algae.

in.the.oceans.and.an.abundance.of.plants.in.the.Devonian, Carboniferous,.and.Permian eras. In.

support.of.this.hypothesis,.the.geological.record.shows.a.drop.in.atmospheric.CO2.at.the.same.time.

as.plants.photosynthesized.

The.O2.partial.pressure.dropped.during.the.Triassic period to.about.15%.(the.CO2.level.rose.

again),.then.after.some.fluctuations.rose.to.about.24%.during.the.Cretaceous/Cenozoic eras.and.

finally.dropped.to.its.modern.level.of.21%.[RSBS.2006;.Palaeos.2002]..Atmospheric.O2.supported.

creatures. with. aerobic. metabolisms. and. permitted. an. ozone. layer. to. form,. protecting. life. from.

mutation-causing.ultraviolet.photons.from.the.sun.

Another.ecologically.important.property.of.certain.cyanobacteria.is.their.ability.to.convert.

(fix).atmospheric.N2 .gas.to.nitrates.and.ammonia,.bioavailable.forms.of.which.aid.growth.in.chlo-rophyll-containing.plants..We.can.also.thank.early.photosynthetic.cyanobacteria.for.supplying.the.

basic.genome.for.the.plant.(eukaryote).chloroplast.[Sato.2002;.Brinkman.et.al..2002;.Hasebe.et.

al..2003].

An.important.theory.is.that.the.chloroplasts.of.plants.originated.through.endosymbiosis.from.

cyanobacteria.with.eukaryotic.cells..Chloroplasts.are.members.of.a.class.of.subcellular.organelles.

known.as.plastids.having.specialized.functions..Raven.and.Allen.[2003].have.commented:.“….plas- tids.contain.DNA,.RNA.and.ribosomes,.supplying.a.structural.and.biochemical.basis.for.non-Men-delian,.cytoplasmic.inheritance.of.plastid-related.characters..Subsequent.molecular.genetic.studies.

have.demonstrated.the.ubiquity.of.plastid.genomes.and.confirmed.that.their.replication,.transcrip-tion.and.translation.closely.resemble.those.of.(Eu)bacteria.”

Molecular.phylogenetic.studies.now.make.it.abundantly.clear.that.the.closest.bacterial.homologs.of.

plastids.are.indeed.cyanobacteria,.supporting.earlier.conclusions.from.the.comparative.biochemistry.

of.photosynthesis..Only.cyanobacteria.and.chloroplasts.have.two.photosystems.and.can.split.water,.to.

make.oxygen,.as.a.source.of.reducing.power..[Quoted.with.permission.]

As. we. have. noted. earlier,. one. of. the. earliest. single-cell. life-forms. to. appear. on. Earth. were. the.

Archaebacteria.. The. Archaea. have. two. phyla:. Crenarchaeota and Euryarchaeoata. This. inter-esting.group.of.prokaryotes.still.has.the.ability.to.live.in.extreme.environmental.conditions;.their.

metabolic.enzymes.are.unique.in.being.able.to.function.at.extreme.temperatures..There.are.over.

250.named.species.of.Archaea. Their.biochemical.adaptations.are.unique..Certain.Archaeans.can.

metabolize.H2,.S,.and.CO2,.materials.found.in.the.early.Earth’s.crust.and.atmosphere.[Kimball.

2004c]..Methanogens.take.in.molecular.hydrogen.gas.and.water.and.metabolize.them.to.methane.

gas.(CH4).and.more.water..A.salt-loving.Archaean,.Halobacterium, contains.bacteriorhodopsin,.a.

purple,.photon-trapping.pigment.that.the.halophile.uses.for.energy..Trapped.photon.energy.pumps.

protons.(H+.ions).to.the.outside.of.the.cell.membrane..When.these.protons.flow.back.inside,.they.

provide.energy.for.the.synthesis.of.ATP,.used.to.power.other.chemical.reactions.in.the.bacterium..

This.important.process.is.called.chemiosmosis [Kimball.2007b,c].

This.is.not.photosynthesis.as.practiced.in.the.chloroplasts.of.higher.plants,.but.a.first.step.in.

trapping.photons.for.energy..The.earliest.Archaebacteria.probably.appeared.on.Earth.over.3.billion.

years.ago.[Armstrong.1997].

Still.another.candidate.for.early.life.on.Earth.is.found.among.the.putative.Nanobacteria,.or.

nanobes..They.were.first.described.by.Folk.[1992,.1993].in.Italian.hot.spring.minerals.as.spheri- cal,.calcified.fossils,.about.25–200.nm.in.diameter..Folk.called.them.Nannobacteria..NASA.sci-entist.Chris.Romanek.had.heard.a.presentation.on.nanobes.by.Folk.and.decided.to.look.for.their.

fossils.in.a.meteorite.of.Martian.origin..He.found.“fossil.nanobes”.embedded.in.the.meteorite,.

which.had.been.radioisotopically.dated.at.about.4.5.billion.years..The.fossils.were.20.to.200.nm.

across..Was.this.evidence.of.microbial.life.on.Mars?.Most.skeptics.replied.that.prokaryotic.life.

as.we.know.it.today.would.require.more.than.a.200-nm.diameter.“cell”.to.contain.all.the.nucleic.

acid,.enzymes,.and.a.few.mitochondria.that.a.living,.minimal,.prokaryote.cell.would.need..Others.

were.not.so.sure..In.1997,.scientists.at.the.University.of.Queensland.discovered.fossil.nanobes.in.

ancient.Australian.sandstones..Then,.Kajander.and.Ciftcioglu.[1998].found.(presumably.living).

coccoid.(spherical).nanobacteria.in.mammalian.blood..The.nanobes.ranged.in.size.from.20.to.

200.nm..They.assumed.that.they.had.discovered.new.living.bacteria.and.named.them.as.Nanao-bacterium sanguineum.

Nanobes.were.subsequently.found.in.(living).human.dental.calculus.and.arterial.plaque..They.

could.be.cultured.and.grown.under.cell.culture.conditions.and.were.shown.to.produce.carbonate.

hydroxyapatite.on.their.“cell”.envelopes..This.mineral.accretion.was.related.to.the.possible.accu-mulation.of.atherosclerotic.plaque.and.renal.calculi.[Kajander.&.Ciftcioglu.1998]..In.optimal.cell.

culture.conditions,.their.doubling.time.was.about.3.days..Similar.to.viruses,.gamma.radiation.did.

not.seem.to.affect.their.viability;.over.1.Mrad.had.no.effect!.Also,.their.growth.could.be.inhibited.

by.tetracycline, 5-flourouracil,.and.cytosine arabinoside.but.not.by.chloramphenicol, rifampin,.

kanamycin, erthromycin,.or.0.1%.sodium azide,.which.inhibits.respiration.[Kajander.&.Ciftcioglu.

1998;.Cisar.et.al..2000].

Two factors.led.other.workers.to.question.the.conclusions.of.Kajander.and.Ciftcioglu..They.are.

the.following:.(1).The.putative.(calcified).bacterial.envelope.was.considered.too.small.to.enclose.

the. necessary. molecular. machinery. to. run. even. a. primitive. prokaryotic. cell,. and. (2). a. DNA.

genome,.RNA,.and.cytoplasmic.protein.from.nanobes.had.not.yet.been.isolated.and.described..

The.size.argument.can.be.rebutted.by.pointing.to.the.soil.ultramicrobacteria.described.by.Bae.et.

al..[1972],.which.have.diameters.as.small.as.about.80.nm..Other.marine.ultramicrobacteria.have.

also.been.described.[Button.et.al..1993]..The.DNA,.RNA,.and.protein.question.is.more.problem-atic,.however.

In.2000,.Cisar.et.al..published.a.stunning.rebuttal.to.the.assumption.that.nanobes.are.“alive.”.

We.quote.their.abstract.in.the.following.text:

The.reported.isolation.of.nanobacteria.from.human.kidney.stones.raises.the.intriguing.possibility.that.

these.micro-organisms.are.etiological.agents.of.pathological.extraskeletal.calcification..[Refs.].Nano-bacteria.were.previously.isolated.from.FBS.[fetal.bovine.serum,.a.culture.medium].after.prolonged.

incubation.in.DMEM.[another.culture.medium]..These.bacteria.[sic].initialized.biomineralization.of.

the.culture.medium.and.were.identified.in.calcified.particles.and.biofilms.by.nucleic.acid.stains,.16S.

rDNA. sequencing,. electron. microscopy,. and. the. demonstration. of. a. transferable. biomineralization.

activity..We.have.now.identified.putative.nanobacteria,.not.only.from.FBS,.but.also.from.human.saliva.

and.dental.plaque.after.the.induction.of.a.0.45-µm.membrane.filtered.samples.in.DMEM..Although.

biomineralization.in.our.“cultures”.was.transferable.to.fresh.DMEM,.molecular.examination.of.decal-cified.biofilms.failed.to.detect.nucleic.acid.or.protein.that.would.be.expected.from.growth.of.a.living.

entity..In.addition,.biomineralization.was.not.inhibited.by.sodium.azide..Furthermore,.the.16S.rDNA.

sequences.previously.ascribed.to.Nanobacterium sanguineum.and.Nanobacterium sp..were.found.to.

be.indistinguishable.from.those.of.an.environmental.organism,.Phyllobacterium mysinacearum,.which.

was.previously.detected.as.a.contaminant.in.PCR..Thus,.these.data.do.not.provide.plausible.support.for.

was.previously.detected.as.a.contaminant.in.PCR..Thus,.these.data.do.not.provide.plausible.support.for.