[soil biology] nucleic acids and proteins in soil volume 8 || horizontal gene transfer by natural...

15 Horizontal Gene Transfer by NaturalTransformation in Soil EnvironmentAnne Mercier, Elisabeth Kay, Pascal Simonet

15.1Introduction

Based on recent data from complete bacterial genome sequences, horizon-tal gene transfer (HGT) would have been a major evolutive force in bacterialevolution. However, even if some bacteria exhibit a particularly high levelof laterally acquired genetic information (Ziebuhr et al. 1999; Ochman et al.2000; Gupta and Maiden 2001; Jain et al. 2002), other bacterial species havedeveloped efficient mechanisms to limit the rate of exogenous DNA acqui-sition in order to better stabilise their genome (Lorenz and Wackernagel1994; Matic et al. 1996; Nielsen 1998). The balance between HGT, whichconfers specific capabilities and gene conservation, which aids in bacte-rial classification, demonstrates the importance of understanding bacterialplasticity (Arber 2000; Woese 2000; Brochier et al. 2002; Daubin et al. 2002;Brown 2003; Kurland et al. 2003). Moreover, this understanding has directapplications in assessing the risks related to the use of genetically modifiedorganisms such as plants, which could become a possible source of trans-forming DNA for environmental bacteria (Nielsen et al. 1998; Bertolla andSimonet 1999). Interest in the fate of transgenes was mainly due to per-sistence of antibiotic resistance genes that were initially used as selectivemarkers for plant construction (Flavell et al. 1992; Scutt et al. 2002), in plantgenomes and its potential transfer to human pathogenic bacteria (Drögeet al. 1998; Normark and Normark 2002). The bacterial origin of these newplant genes could help them overcome the bacterial genetic barriers thathad prevented all but a few rare plant genes being transferred from plantsto bacteria (Brown and Doolittle 1999; Brinkman et al. 2002). In addition,the risk of transgene transfer would increase when transplastomic plantsare considered (presence of the transgene in the chloroplast genome) dueto the higher copy number that is increased from 10 in traditional andnuclear-modified transgenic plants to more than 10,000 for transplastomicplants (Daniell et al. 1998).

Anne Mercier, Elisabeth Kay, Pascal Simonet: Ecologie Microbienne, UMR 5557, Uni-versité Lyon I, 43 bd du 11 Novembre 1918, 69622 Villeurbanne, France, E-mail:[email protected]

Soil Biology, Volume 8Nucleic Acids and Proteins in SoilP. Nannipieri, K. Smalla (Eds.)© Springer-Verlag Berlin Heidelberg 2006

356 A. Mercier et al.

HGT in bacteria has been investigated at various levels including insilico analyses, with appropriate bioinformatics tools to infer transferredgenes in complete genome sequences (Eisen 2000; Garcia-Vallvé et al. 2000,2003; Brown 2003; Daubin et al. 2003). Gene-transfer mechanisms inclu-ding natural transformation, conjugation and transduction have also beenstudied at the genetics and physiological levels on some bacterial models(Amabile-Cuevas and Chicurel 1992; Lorenz and Wackernagel 1994; Dub-nau 1999). Microcosms and open environment based experiments havebeen developed to determine the occurrence of gene transfer in situ andits potential impact on microbial community and ecosystems (Lorenz andWackernagel 1994; Nielsen et al. 1998; Davison 1999; Dröge et al. 1999; Paul1999; Timms-Wilson et al. 2002).

Critical questions related to assessing the gene-transfer events betweenbacteria and the risk in using transgenic organisms include determiningwhere gene transfer could occur in the environment, which molecularmechanisms are involved, and how they are regulated in situ by the bacteriathemselves and also by environmental factors.

15.2Mechanisms of Horizontal Gene Transfer

In bacteria, three mechanisms, including conjugation, transduction andnatural transformation, have been reported to be involved in HGT (Yinand Stotzky 1997; Ochman et al. 2000). Conjugation (see Chap. 14) andtransduction promote transfer of genetic elements like plasmids, bacterio-phages, genetic islands, transposons and integrons. These accessory ele-ments are important vectors for the spread of genes coding for pathogenictraits, resistance to heavy metals or antibiotics among bacteria (Dobrindtand Hacker 1999, 2001; Rowe-Magnus and Mazel 2001). Some of these ele-ments, such as conjugative plasmids or transposons, contain all or most ofthe genes required to promote their own transfer to bacteria belonging tothe same or different species.

In contrast, natural transformation, which is the active uptake by com-petent bacteria of extracellular DNA present in their surrounding environ-ment, requires coordinated functions encoded by a set of genes distributedthroughout the genome of recipient bacteria (Lorenz and Wackernagel1994; Friedrich et al. 2001). Products of these genes are involved in thefour interconnected steps of the natural transformation process, includingcompetence development, DNA binding, DNA uptake and the inheritableintegration of the incoming DNA (Lorenz and Wackernagel 1994; Dubnau1999; Chen and Dubnau 2003). Natural transformation-mediated gene ac-quisition and illegitimate or homologous recombination-based genomic

15 Horizontal Gene Transfer by Natural Transformation in Soil Environment 357

integration of incoming DNA into the host genome were demonstrated tooccur both among bacteria belonging to the same species and to phyloge-netically less related taxa (Lorenz and Wackernagel 1994; Kriz et al. 1999;Sikorski et al. 2002).

While transduction and natural transformation were often regarded asecologically irrelevant in soil environments (Davison 1999), conjugationwas considered as the most efficient gene-transfer mechanism occurringin situ. It was also the easiest mechanism to investigate by the use of micro-or mesocosms in which conjugative plasmid transfer frequencies betweendonor and recipient bacteria could be determined (Sengelov et al. 2000,2001; de Lipthay et al. 2001; Desaint et al. 2003). According to these studies,several soil environments were recognised as being “hot spots” for HGT incomparison to the bulk soil including the rhizosphere (soils surroundingplant roots), the spermosphere (soils surrounding germinated seeds) and,finally, the “residuesphere” (interface between decaying plant material andsoil matrix; Van Elsas et al. 1988; Troxler et al. 1997; Sengelov et al. 2000,2001; de Lipthay et al. 2001; Nielsen et al. 2001).

15.3In Situ Regulation of Natural Transformation in Bacteria

Several processes related to the bacteria themselves or to their environmentappear to limit the occurrence of transformation-mediated genetic trans-fer. The number of bacteria described as being naturally transformable isrelatively low, thus limiting the potential impact of the phenomenon on themicrobial community (Lorenz and Wackernagel 1994; Davison 1999). Intransformable bacteria, the uptake of DNA is submitted to a series of filtersby several successive mechanisms that decrease success rates. In some com-petent bacteria, transformation is restricted to DNA from the same species,i.e. to DNA that contains species-specific “uptake signal sequences” re-quired in order that the DNA binds to the cell wall prior to uptake (Smithet al. 1999). Although restriction and modification systems recognise andcleave double-stranded DNA, they can also degrade single-stranded DNAproduced during natural transformation (Ando et al. 2000; Aras et al. 2002;Berndt et al. 2003). The last step of genetic transformation is the stable in-heritance of the entering DNA either by autonomous replication for a plas-mid molecule or by recombination-mediated genomic integration of linearDNA (de Vries and Wackernagel 2002, Meier and Wackernagel 2003a).

Efficiency of integration by homologous recombination depends on thesize of the DNA and sequence divergence between donor and recipient DNAregions (Strätz et al. 1996; Demanèche et al. 2002). Two main antagonisticsystems, the mismatch repair system (MRS) and the SOS system, are


involved in the regulation of heterologous DNA integration (Matic et al.1996; Taddei et al. 1997; Nielsen 1998; Young and Ornston 2001). Bacte-rial genome stability depends on the expression level of these two systemswith the MRS acting as a strong but flexible genetic barrier to prevent anyuncontrolled genetic drift due to integration of heterologous DNA.

Occurrence of natural transformation in the environment was also ques-tioned due to adverse conditions encountered by DNA and bacteria in situ.Soil conditions were thought to be physically, chemically and enzymaticallyincompatible with long-term persistence of free DNA. In addition, severalstudies failed to demonstrate that bacteria could develop competence insitu (see below).

15.4Natural Transformation: An Unexpected WidespreadGene-Transfer Mechanism in Bacteria?Extracellular DNA present in the vicinity of bacteria is used by most bacte-ria in situ as a source of carbon and energy for supporting bacterial growth.In addition this DNA can also be used for genome evolution relevant func-tions such as DNA repair (Michod et al. 1988; Mongold 1992; Redfield1993; Solomon and Grossman 1996; Redfield et al. 1997; Finkel and Kolter2001). Interestingly, DNA consumption in non-competent bacteria suchas Escherichia coli and some other proteobacteria involves enzymes thatare homologous to proteins involved in the DNA-uptake function of com-petence in naturally transformable bacteria (Finkel and Kolter 2001). Theevolutive interest in these functions is further supported by the detection ofhighly homologous modules composed of DNA-uptake genes in distantlyrelated bacteria suggesting possible acquisition of these modules by HGTand their integration at different genomic loci in the host bacterial genomes(Friedrich et al. 2001).

The list of bacteria known to possess a genetically encoded naturaltransformation machinery seems very limited in comparison to the hugebacterial diversity. However, these bacteria encompass a wide range ofphysiological capabilities: photolithotrophy, chemolithotrophy, heterotro-phy, and methylotrophy (Table 15.1). While most of the known naturallytransformable bacteria are mesophiles, some are thermophiles, and the listincludes saprophyte bacteria as well as plant and animal symbionts andpathogens (Lorenz and Wackernagel 1994). However, the transformationpotential of soil bacteria remains almost totally unknown; most isolatesfrom worldwide culture collections having never been tested for compe-tence development under in vitro or in situ conditions (Demanèche etal. 2001c). Moreover, the competence development property is not sys-


Table 15.1. Non-exhaustive list of naturally transformable bacteria isolated from soil envi-ronment

Species present in soil environment References

Natural transformable bacteria (competence regime identified)Photolithotrophic Synechocystis sp. PCC 6803a Yoshihara et al. 2001Chemolithotrophic Thiobacillus thiopuransb Yankofsky et al. 1983Heterotrophic Acinetobacter calcoaceticusa Palmen et al. 1993

Azotobacter vinelandiib Page and Grant 1987Bacillus subtilisa Dubnau 1991Deinococcus radiodurans Fuchs et al. 1994Mycobacterium smegmatis Norgard and Imaeda 1978;

Bhatt et al. 2002Pseudomonas stutzeria Carlson et al. 1983; Meier et al.

2002Ralstonia solanacearum Bertolla et al. 1997Thermus thermophilusa (andThermus spp.)

Koyama et al. 1986; Friedrichet al. 2001, 2002

Methylotropic Methylobacteriumorganophilumb

O’Connor et al. 1977

Archaebacteria Methanobacteriumthermoautotrophicumb

Worrell et al. 1988

Clinical pathogenic spp. Campylobacter jejunia Wiesner et al. 2003Campylobacter coli Wang and Taylor 1990Helicobacter pyloria Hofreuter et al. 2000; Smeets

and Kusters 2002Legionella pneumophilaa

(serogroup 1)Stone and Kwaik 1999

Moraxella spp.a Juni et al. 1988; Luke et al. 2004

Suspected natural transformable bacteria (competence regime non-identified)Agrobacterium tumefaciensd Demanèche et al. 2001cEscherichia colic Claverys and Martin 2003Lactococcus lactisc Bolotin et al. 2001Listeria monocytogenesc Claverys and Martin 2003Pseudomonas fluorescensd Demanèche et al. 2001c

a Competence regime identified and completed by molecular analyses of the naturaltransformation machinery

b No recent publication about natural transformation of the species consideredc In silico detection of a natural transformation machineryd In situ evidence of natural transformation capability

tematically shared by all representatives belonging to the same species, andtransformation frequencies can vary up to four orders of magnitude amongtransformable isolates of the same bacterial species (Sikorski et al. 2002).

The number of transformable bacteria (44) proposed 10 years ago byLorenz and Wackernagel (1994) has increased significantly with data from


complete genome sequences and experimental studies (Claverys and Mar-tin 2003; Davidsen et al. 2004). Genes encoding homologues of the DNA-uptakemachinery componentsweredetected innumerousbacteria, includ-ing Lactococcus lactis, Streptococcus pyogenes, and E. coli, for which naturalcompetence development had not been reported previously (Bolotin et al.2001; Claverys and Martin 2003). Thus, these bacteria might also undergoan active and genetically encoded transformation process.

In silico detection of competence sequences in bacterial genomes moti-vates new experimental studies in order to determine if the related genesare still functional and which conditions are necessary for their expression(Claverys and Martin 2003). Such studies could rapidly lead to a signifi-cant extension of the list of naturally transformable bacteria and to a betterunderstanding about genesis, evolution and the spread of the genetic trans-formation mechanism among bacteria and its role in adaptation.

15.5Bacterial Competence Development in Soil

The level to which a bacterial population is modified by natural transfor-mation depends on natural transformation frequencies directly related tothe ability of bacteria to take up DNA. Natural transformation has beendetected in various environments including seawater and marine sedi-ments (Paul et al. 1991; Paul 1999), plant tissues (Bertolla et al. 1999; Kayet al. 2003) and soil (Lorenz and Wackernagel 1994; Nielsen et al. 1997,2000a,b; Dröge et al. 1999). Most of these studies included the use of natu-rally transformable bacteria inoculated in microcosms or mesocosms withthe objective to simulate natural conditions demonstrating that competentbacteria can take up extracellular DNA in situ.

Bacteria such as Bacillus subtilis, Acinetobacter spp. and Pseudomonasstutzeri were selected as models on the basis of criteria related to theirtransformation potential in vitro (Lorenz and Wackernagel 1994; Kay et al.2002a,b; de Vries et al. 2001).

Naturally transformable bacteria exhibit significant differences in com-petence development (Solomon and Grossman 1996). Competence is ex-pressed constitutively in few bacteria but this state is more often relatedto an active physiological state corresponding to the exponential growthphase under in vitro conditions (Lorenz and Wackernagel 1994; Solomonand Grossman 1996). Other bacteria require accumulation of a compe-tence factor in the surrounding medium (Solomon and Grossman 1996).For those bacteria, only cells at the stationary phase become competent(Lorenz and Wackernagel 1994). These differences between bacteria can be


explained by their need to adapt to specific environmental niches depend-ing on their location in soil microhabitats.

The biotic and abiotic conditions of the different soil microhabitats, suchas competition with other bacteria, predation, pH, moisture level, concen-tration of mono- and bivalent cations, and nutrient input, are known toaffect the natural transformation process (Timms-Wilson et al. 2002). Soilsare often described as being more refractory than marine environmentsfor bacterial competence development due to the matrix heterogeneity andthe oligotrophic conditions which lead bacteria to survive in a state of dor-mancy (Lorenz and Wackernagel 1994; Nielsen et al. 1997). For example,Acinetobacter sp. strain BD413, which exhibits a particularly high transfor-mation frequency in vitro (Palmen et al. 1993), did not develop competencenaturally in soil (Nielsen et al. 1997). This competence state was also lostrapidly when Acinetobacter spp. competent cells prepared in vitro weresubsequently inoculated in oligotrophic soils (Nielsen et al. 1997). Severalapproaches for overcoming these limitations have been studied. Nutrientamendment and a high phosphate level led to Acinetobacter sp. BD413 com-petence in soil. In addition, transformation frequencies were also found tobe higher in a silt loam than in a loamy sandy soil (Nielsen et al. 1997).

Nielsen and Van Elsas (2001) studied Acinetobacter sp. BD413 bacterialcompetence stimulation in sterile and non-sterile soil after addition of var-ious organic compounds that are found naturally in the rhizosphere of cropplants. Mixtures of organic and amino acids such as acetate, lactate andalanine, sugars, mainly glucose, and the high-P salts have a pronouncedeffect on natural transformation frequencies of Acinetobacter sp. BD413 innon-sterile soil. Many compounds exuded into the rhizosphere by agricul-tural plants are able to increase bacterial metabolic activity (Kroer et al.1998) and stimulate competence development and natural transformation(Nielsen and Van Elsas 2001).

Heterogeneity of the soil matrix must be seriously consideredbefore con-cluding that soil environments are unable to provide favourable conditionsfor natural competence development. Bacteria can develop in biofilms onroots, decaying organic matter, and even mineral particles, thus improvingthe adapted conditions for DNA-transfer events including natural trans-formation (Nielsen et al. 1998; Paul 1999). Spatial distribution of bacteriaat the microhabitat scale level is currently under investigation with specificand sensitive molecular methods, which should provide new data about theprobabilities of contact between bacteria and extracellular DNA or otherbacteria (Ladd et al. 1996; Grundmann and Debouzie 2000; Dechesne et al.2003; Vogel et al. 2003).


15.6Gene Transfer in the Environment by Alternate GeneticTransformation-Related Mechanisms?

One isolate belonging to the genus Pseudomonas fluorescens could notbe naturally transformed in vitro under a wide range of conditions, butprovided transformants when inoculated in sterile or non-sterile soil in thepresence of plasmid DNA (Demanèche et al. 2001c). Similar results wereobtained with E. coli that formed transformants in river water (Baur et al.1996), foodstuff (Bauer et al. 1999), urine (Woegerbauer et al. 2002) and onagar plates (Tsen et al. 2002). These results indicate that in situ biotic andabiotic conditions regulate DNA uptake in a wider range of bacteria thanthose already referenced as being naturally transformable. Whether thesebacteria undergo an active DNA-uptake process similar to that relatedto competence development or are subjected instead to a passive DNAentry due to chemically or physically mediated cell wall and membranepermeabilisation is still unclear. Other environmental conditions, suchas those related to lightning discharges, can lead to the natural electro-transformation of bacteria in soil (Demanèche et al. 2001a; Cérémonie et al.2004). Lightning-related electrical parameters, including current injectionand electrical fields, are the same (when considered at the same spatialscale) as those delivered by electroporators that are used to transforma wide range of bacteria in the laboratory (Demanèche et al. 2001a).

15.7Persistence of Extracellular DNA in Soil

A key factor of bacterial transformation frequency in soil is the availabilityof non-degraded and biologically active DNA after its release from dead(or living) cells. Availability means that extracellular DNA has to escapechemical and enzymatic degradation until a contact is established witha transformable bacterium. Most of the initial work that aimed at studyingthe occurrence of natural transformation-mediated gene transfer in soilfocused on the detection of extracellular DNA in soil, and, thus, led to thestudy of DNA-degradation kinetics and mechanisms.

Significant amounts of non-degraded DNA were detected in naturalsoils (Frostegård et al. 1999). This confirmed results of microcosm-basedexperiments in which purified genomic (chromosomal or plasmidic) DNAfrom bacteria (Romanowski et al. 1992; Widmer et al. 1996; Gebhard andSmalla 1999), bacterial lysates (Nielsen et al. 2000a) but also plant DNA,ground plant material (Widmer et al. 1996), plant leaves (Widmer et al.


1997), decaying plant material (Ceccherini et al. 2003) and pollen (Meierand Wackernagel 2003b) were inoculated into soil before re-extraction.

Persistence of most extracellular DNA in soils was due to a reversible ad-sorption onto soil components including sand particles, humic compoundsand particularly clay minerals, such as montmorillonite, illite, and kaolin-ite (Paget et al. 1992; Crecchio and Stotzky 1998; Demanèche et al. 2001b;see Chap. 7). The efficiency of the DNA-adsorption process depended onthe size of the DNA molecules and their linear, open circular or supercoiled conformations (Gallori et al. 1994; Poly et al. 2000; Demanèche etal. 2001b). Adsorption efficiency was also affected by soil parameters suchas pH, temperature, concentration and cation valency (Romanowski et al.1991; Khanna and Stotzky 1992; Lorenz and Wackernagel 1994). In addi-tion to a specific protective effect of DNA adsorption, nucleases are alsoadsorbed onto clay particles leading to a physical separation between en-zymes and their DNA substrate, thus increasing the protective effect ofadsorption (Demanèche et al. 2001b). Finally, interactions between celldebris, including membranes and cytoplasmic residues, and DNA con-tributed to the prevention of rapid biological inactivation of DNA (Nielsenet al. 2000a).

Due to the protective effect of adsorption, the turnover of a significantpart of extracellular DNA released into soil by various organisms includinganimals, plants, fungi or bacteria would be quite slow; this slow rate ex-plains the positive detection by polymerase chain reaction (PCR) of DNAafter its incubation for months and even years in soil (Paget et al. 1992; Ro-manowski et al. 1993; Widmer et al. 1996, 1997; Gebhard and Smalla 1999).However, hybridisation or PCR-based detection of DNA did not provideany information about the biological potential (transforming ability) thatthe persisting DNA could maintain throughout its incubation. Preliminaryresults based on inoculation of pure DNA in soil indicate that biologicalactivity of DNA for competent bacteria decreased rapidly, in only a fewhours (Gebhard and Smalla 1999), demonstrating a discrepancy betweenthe physical stability and the functional significance of chromosomal DNAin soil (Nielsen et al. 2000a).

15.8Development of Methods To Investigate Gene Transfer

Most of the in situ transformation experiments conducted recently haveinvolved inoculation into sterile or non-sterile soil samples of donor DNA(plasmidor chromosome) incombinationwithbacteria already ina compe-tent state. Broad host range plasmids were used allowing the transformingDNA to be replicated in various bacteria (Lorenz and Wackernagel 1994;


Timms-Wilson et al. 2002). When chromosomal DNA from bacteria orplants was used, specific constructions were developed in which one ortwo easily selective marker genes were flanked by homologous sequencesin order to promote homologous recombination-mediated integration inthe recipient host (de Vries et al. 2001). Detection of transformants re-quired expression of the newly acquired marker genes to confer antibioticresistance allowing the recombinant clones to grow under selective pres-sure. PCR and/or DNA hybridisations were used to confirm the presence ofdonor-specific DNA sequences in the recombinant clones. A derivative ofthis approach, the marker rescue system, was proposed in which donor andrecipient DNA contained the same marker gene with the only exception thatexpression was inactivated in the recipient strain by deletion of the centralpart of the gene. Expression was rescued after transformation with donorDNA and homologous recombination between donor and recipient DNAbasedon the remaining sequencesof themarker gene (deVries andWacker-nagel 1998; Gebhard and Smalla 1998; Nielsen et al. 2000b; Kay et al. 2002b;de Vries et al. 2003; Meier and Wackernagel 2003b). Moreover, the deletedregion was used as a probe to demonstrate that the rescued phenotypewas due to the expected transfer event. The rescue marker systems wereused to study the potential of antibiotic resistance genes from geneticallymodified plants to be transferred to bacteria. The system was adapted withthe nptII gene used in nuclear-modified potatoes and sugar beet plants andthe aadA gene from transplastomic (the chloroplast genome was modified)tobacco plants that were cloned after deletion in Acinetobacter spp. and/orPseudomonas stutzeri (Nielsen et al. 2000b; Kay et al. 2002; de Vries et al.2003). Transformation with plant DNA restored a complete and functionalnptII or aadA gene conferring resistance to kanamycin and spectinomycin-streptomycin, respectively. Applications of these specific and sensitive ap-proaches included determination of the transformation potential of plantDNA which persisted in soil. In most cases, total DNA was extracted fromsoil and used to transform the recipient bacteria in vitro (Timms-Wilsonet al. 2002; Meier and Wackernagel 2003b). Another application deals withthe use of the recipient strain in situ (soil or plant) which requires thesubsequent and specific isolation of transformants. This approach remainsproblematic in soil environments which contain a significant proportion ofantibiotic-resistant indigenous bacteria.

If the detection of transformation-mediated gene-transfer events thatwould have occurred naturally in situ among indigenous soil bacteriaremains extremely difficult due to a lack of effective marker sequences,there are some possibilities for detecting bacteria that would have acquiredthe genes from transgenic plants. In numerous cases, these plants containmarker genes, such as antibiotic-resistance genes, which could facilitate theselection of bacteria which would have acquired these genes. This approach


requires, however, selecting between the recently generated transformantsand indigenous bacteria naturally fitted with the same gene. This can bedone by detecting sequences specific to the plant construction that flank themarker gene and that could have been co-transferred to bacteria. Presenceof these transgenic plant-specific synthetic DNA sequences in soil bac-teria could demonstrate that plant-bacteria DNA transfer had occurred.However, to date there is no report of the isolation of bacteria in whichplant DNA sequences can be detected from soils cultivated with transgenicplants.

15.9Gene Transfer by Natural Transformationfrom Transgenic Plants to Bacteria – A Possible Event?Plant transgenes may behave like any other naturally occurring genes inthe soil environment but they often differ in several ways from native geneswith respect to their likelihood of gene transfer, expression in the new host,and selection. A successful (and detectable) gene transfer from transgenicplants to bacteria requires several events to occur successively, thus justi-fying that each of the steps was investigated separately to determine thelimiting factor. During the first step, DNA must be released by the plant.A study by Ceccherini et al. (2003) demonstrated that most of the DNA wasdegraded during the plant decaying process. Only a minor fraction of theplant DNA was susceptible to a release into the soil. These data indicate thatendophyte bacteria, including pathogens and other colonising bacteria, arethose forwhichexposure toplantDNAwouldbe themost significant (Kayetal. 2002a). In addition to a favoured access to plant DNA, plant tissues couldbe environments in which bacteria easily reach competence. However, themain barrier to gene transfer would not be a physical one (degradationand/or unavailability of the DNA) or a physiological one (inability of bac-teria to take up DNA), but a genetic barrier that prevents exogenous DNAbeing integrated in the recipient genome of bacteria. The results of Kay etal. (2002b), which demonstrate that a marker gene from a transplastomicplant could be transferred in planta only to bacteria possessing the appro-priate homologous DNA sequences, confirm the fundamental role of therecombination step in regulating gene transfer. However, transfer of planttransgenes with a low level of homology with bacteria genomes cannotbe excluded considering that short homology sequences can act as anchorsequences to promote illegitimate recombination of longer DNA fragments(de Vries et al. 2004). Although these events occur at low frequency, theyconfirm that the exchange of DNA between distantly related organismsincluding plant and bacteria is theoretically possible.


15.10Concluding Remarks

Environmental microbiologists have developed numerous studies with theobjectives of providing direct and experimental evidence of the impactof horizontal gene transfer (HGT) events on bacterial genome structure.In silico analysis of complete genome sequences has led to proposals ofrelatively high HGT rates. However, in spite of these efforts, there is stilla discrepancy between the two approaches, and additional experimentalstudies are necessary to confirm the actual role of these mechanisms incomplex environments such as soil or sediment. Location, density, andmetabolism of bacteria in soil microenvironments are still almost unex-plored, and their ability to develop competence is unknown. For example, ifplant DNA was detectable in soils for years (Paget et al. 1992; Gebhard andSmalla 1999), there was no clear evidence that this DNA could be still in-volved in a transformation process with bacteria. However, two points mustbe considered carefully when addressing HGT by natural transformationin soil environments. The first is that studying genetic information transferbetween indigenous bacteria remains very difficult because of the lack ofmarker genes that could allow differentiation between recipient and re-combinant bacteria. Most studies that provided the available data involvedinoculation of donor or recipient bacteria but such approaches are knownto simulate poorly what might occur with indigenous bacteria which donot colonise the same soil microniches as inoculated bacteria. This is whytransgenic plants with marker genes inserted in their genomes have to beconsidered as invaluable and perhaps the only model for studying howgenetic information can be exchanged naturally between living organisms.

The second point is that experimental studies were conducted at themacroscale level without considering the spatial distribution of bacteria atthe microscale level which is the level at which bacteria interact in soil. Toolsneed to be adapted to gene-transfer potentials in the few spots colonisedby bacteria considering that 108 cells per g of soil occupy only 0.1 mm3 ofthe 500 mm3 of pores in 1 cm3 of soil (Pallud et al. 2004).

Acknowledgements. We thank Timothy M. Vogel for his comments andcritical reading of the manuscript.

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