University of Groningen
Psysiological and genetic diversity of the purple nosulfur bacterium Rhodopseudomas
palustris
Oda, Yasuhiro
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Oda, Y. (2002). Psysiological and genetic diversity of the purple nosulfur bacterium Rhodopseudomas
palustris Groningen: s.n.
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Summary and concluding remarks
and hence,they
Microorganismscan readily adaptthroughvariousevolutionaryprocesses
residein a vast array of natural environmentsand sustainvarious biogeochemicalcycles.
bacterialdiversity.
Thus,understanding
thesecycleshas hingedon our ability to characterize
of bacterialdiversity at
Severalmoleculartechniqueshave been used for the assessment
differentlevels of resolution(i.e., genus,speciesor strain).Amongst these,comparisonsof
small subunit ribosomal RNA (165 rRNA) sequencesand short oligonucleotidesthat
hybridizeto specificregionsof rRNA molecules(e.g.,fluorescentin situ hybridization,FISH)
have become among the most powerful tools to assessthe phylogeneticand functional
(Amannet al.,1995; Weisburget al., 1991;Woese,1987).The
diversityof microorganisms
actualabundanceand spatialdistributionof many bacterialspeciesin natural environments
havebeenextensivelystudiedby use of the FISH technique(for review, see Amann eÍ a/.,
1995).However,to date,little is known aboutthe abundanceand spatialdistributionof purple
nonsulfurbacteriain naturalenvironmentsdespitethe fact that this group of bacteriais widely
distributed,and plays an important role in anaerobicnutrient cycles in many environments
(Chapterl). The natural habitatsof purple nonsulfur bacteriaare subjectto substantialand
frequentchangesin environmentalconditions(e.g.,oxygen levels and light penetration)and
thesewill influencethe growth rate and physiologicalstateof thesebacteria.The sensitivityof
FISH using 165 rRNA oligonucleotideprobes is dependenton the cellular abundanceof
rRNA, and thereforeon the cellular growth ratesof purple nonsulfurbacterialpopulationsin
naturalenvironments.The findings presentedin Chapter 2 demonstratedthe inf'luenceof
differentmodesof growth and starvationon FISH in pure culturesof the purple nonsulfur
palustrus.This study showsthat the detectabilityof individual
bacteriumRhodopseudomonas
cells using the FISH techniquewas strongly influencedby their physiologicalhistory and
cunent physiologicalstate. Interestingly,there were significant differencesin metabolic
activity and cell viability of Rps. palustis that had been grown and starved under oxic
conditionsin the dark, or under anoxic conditions in the light. The cell viability of Rps.
palustriscellsgrown and starvedunderanoxic conditionsin the light remainedrelativelyhigh
for a longerperiod of time as comparedto cells that had beengrown and starvedunder oxic
conditionsin the dark, or cells grown under anoxic conditionsin the light and starvedunder
anoxic conditions in the dark. This implies that light serves as a source of energy for
maintainingcell viability in the absenceof an externalgrowth substrate.
Differencesin the physiological characteristicsof individual microorganismstypically
Suchdifferencesarise
resultfrom geneticdifferencesbetweenthe individual microorganisms.
throughevolutionaryprocessesthat selectfor variantswith improved fitness under specific
conditions.Giventhat many environmentsare carbon-limited,mutationsthat conferthe ability
to metabolizea xenobiotic compound can confer a strong selectiveadvantage.This may
accountfor the occurrenceof microbial speciesable to metabolizeanthropogeniccompounds
that haveonly recentlybeen introducedto the environment.Many microbial speciesable to
degradexenobiotic compounds have been isolated from highly polluted environments
(Henicket al.,1991;Leahyand Colwell, 1990;Monis et a\.,1992:van der Meer et al.,1998;
Wu and Wiegel, 1997). While aerobic degradationof xenobiotic compounds has been
extensivelystudied, little is known about the anaerobic mineralization of xenobiotic
compounds.This might be due to the fact that such anaerobicbacteria are not easily
cultivated,becausethey are rare in naturalenvironments,or becausetheseactivitiesare only
expressed
underspecificconditions.
ll0
Severalstudiesin the past suggestedthat purple nonsulfurbacteriaare likely to play an
importantrole in mineralizationof halogenatedcompoundsin naturalenvironments(Eglandet
a\,,2001; Kamal and Wyndham, 1990;McGrath and Harfoot, 1991 Montgomeryand Vogel,
1992: van der Woude et al., 1994). To better understandthe potential of purple nonsulfur
ofRps. palustrisand the selection
bacteriato degradesuchcompounds,the adaptiveresponses
of novel metabolic ability in this specieswere investigated(Chapter 3). All of the Rps.
palttstris strainstestedwere originally able to metabolize3-chlorobenzoate
if benzoatewas
providedas a co-substrate.
However,after long-termexposureto 3-chlorobenzoate
the strains
acquiredthe ability to metabolizethis compound as the sole carbon source.The findings
presentedin Chapter3 clearly demonstratethat adaptivemutationsthat expandthe ability of
Rps. palustri,s to metabolize xenobiotic compounds commonly and reproducibly occur
althoughthe mechanismsresponsiblefor the adaptivechangesremainunknown.The frequent
occuÍrenceof suchadaptivemutations,may well accountfor the metabolicversatilityof Rps.
palustris that is widely recognized(Gibsonand Harwood,1995; Harwoodand Gibson,1988;
Sasikalaand Ramana,1998).
Genetic variation is a prerequisiteof biological evolution. The amount and kind of
variation found in populations apparently differs depending on the life history of
and differencesin selective
microorganisms,
on the frequencyof variousmutagenicprocesses,
pressuresimposedby various habitats.Hence,many speciesof bacteriain the environment
particularlyin soil, havebeenshownto composeofgeneticallydistinctand diverseclonesthat
are well adaptedto local conditions(Cho and Tiedje, 2000; Fulthorpeet al., 1998;Istocker
et al.,1996;MaynardSmither al., 1993;McArthuret al.,1988;Wiseet al.,
al.,1992;Johnsen
1995). In Chapter 4, the phenotypic and genetic characteristicsof Rps. palustris strains
originating from various ecological sourceswere reported.While all strains studiedhad
certain phenotypiccharactersin common, including the ability to metabolizebenzoateand
there were also significant differencesamong the strains
degrade2- and 3-chlorobenzoate,
(Table I in Chapter4). Phylogeneticand geneticcharacterization
of
basedon genesequences
(RubisCO)genes,as
l65 rRNA and form II ribulose 1,5-bisphosphate
carboxylase/oxygenase
well as rep-PCR genomic DNA fingerprinting revealed differences among the strains.
Interestingly,all analysesshowedthe existenceofthree distinctclades.Thesecladesmay have
developed through adaptive evolution of Rps. palustris clones to prevailing specific
environmentalconditionsand this may representan incipientsympatricspeciationevent.
While the results presentedin Chapter 4 demonstratedthe existenceof significant
phenotypicand genotypicdiversity among strainsof Rps.palustris, the apparentextent of
diversity within this speciesmay be distortedby the fact that the strainsusedin this study
were isolated from selective enrichment cultures (Dunbar et al., 1997). To expand our
knowledgeof the extent of diversity within speciesof purple nonsulfurbacteria,Chapter5
of 128 strainsisolated
describedthe phylogenetic,genotypic,and phenotypiccharacteristics
by direct plating of sedimentsamplestaken from 2 ecologicallydistinct sites,Haren and De
Biesbosch,The Netherlands.Strikingly, BOX-PCR genomic DNA fingerprintingrevealed
there were 60 distinct genotypes.Analysesof 165 rRNA gene sequencesof representative
from each genotypeshowed that there were 5 and 8 different speciesof purple nonsulfur
bacteriafrom the Haren and the Biesboschsites,respectively.At the Haren site, Sl% of the
isolates were identified as Rps. palustris, whereas clones of Rhodoferaxfermentans were
numerically dominant at the Biesboschsite and comprised46Voof the total isolates.The
genomic fingerprintsof Rps. palustris strainsfrom the 2 samplingsites were significantly
different. None of the genotypesfound in the Haren site was found in the Biesboschsite.
Theseresultsimply that certain strainsmay be endemicto eachsamplingsite. The resultsof
the study also show that the genotypic diversity within the species Rps.palustis at the
Biesboschsite was significantly higher than that of the Haren site. If as postulatedby
McArthur et al. (1988),the geneticdiversity of speciesincreaseswith habitatvariability, our
findingsof significantdifferencesin genotypicdiversitybetweenthe 2 samplingsitessuggest
that the Biesboschsite exhibitsa greaterhabitat variability than the Haren site. Surprisingly,
not all strainsof Rps.palustris isolatedfrom thesesitescould degradebenzoatein spiteof the
fact that this trait is reportedlycharacteristicof the species(Gibson and Harwood, 1995;
Imhoff andTriiper, 1992).
Studiesof bacterial biogeographyare needed to better understandcurrent population
structuresand their evolutionary history. However, to date, little is known about the
biogeography
of free-living bacteriain naturalenvironments.This is in contrastto the many
plant-, animal-, and human-associated
bacterial species(see Chapter l). In Chapter 6, the
biogeographyof Rps. palustris strains isolated from aquatic sedimentswas investigated.
Sedimentsampleswere collectedalong a linear l0 m transect,and 30 clonesfrom each of 5
samplinglocationswere characterizedby BOX-PCR genomic DNA fingerprinting.Cluster
analysisof 150 genomicfingerprintsshowedthere were 4 major genotypesof Rps.palustris
(A, B, C, and D, Fig. 2 in Chapter 6) and each genotypeexisted in defined patternsof
distribution.The differencesin genomicfingerprint pattemsamongthe 4 major genotypesof
Rps.palustris were accompaniedby the differencesin phenotypic characteristics.These
resultsindicatethat each genotypeis a distinct clade and functional differencesthat have
ecologicalsignificancemay exist among theseclonal lineages.Moreover,the clonal lineages
observedalong the sampling transectmay representecotypesthat have diverged through
sympatricspeciation.Morisita-Hom similarity coefficients(Stiling, 1999) that comparethe
numbersof common genotypesfound in pairs of samplinglocationsshowedthat there was
substantialsimilarity between locationsthat were I cm apart (Crran= 0.97), but there was
almostno similarity betweenlocationsthat were > 9 m apart(Cvlr < 0.25).Thesecalculations
showeda gradualdecreasein similarity among the 5 locationsas a function of distance,
indicatingthat natural populationsof R2s. palustris are assemblages
of geneticallydistinct
ecotypesandthe distributionofeach ecotypeis patchyin its distribution.
While the outcomeof Chapters4, 5, and 6 yielded a significantinsight into the genotypic
and phenotypicdiversity, and biogeographyof natural populationsof Rps.palustris, further
is neededto answerthe ecologicalsignificanceof the observedpatternsof genotypic
research
andphenotypicdiversityamonglocal populationsofRps. palustris.
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