DENOMINATION DU MEDICAMENT Myozyme® 50 mg poudre pour solution à diluer pour perfusion. 2. COMPOSITION QUALITATIVE ET QUANTITATIVE Un flacon contient 50 mg d’alpha alglucosidase. Après reconstitution, la solution contient 5 mg d’alpha alglucosidase* par ml et après dilution, la concentration varie de 0,5 mg à 4 mg/ml. *L’α-glucosidase acide humaine est produite par
Inbi.canterbury.ac.nzProblems in monitoring horizontal gene transfer in
field trials of transgenic plants
Jack A Heinemann1,2 & Terje Traavik2
Transgenic crops are approved for release in some countries,
failed3,17–19. HGT from a transgenic organism into the genome of a while many more countries are wrestling with the issue of
recipient organism has been detected in the environment, but not .com/naturebiotec
how to conduct risk assessments. Controls on field trials often
without the use of recipient bacteria carrying special constructs (e.g., include monitoring of horizontal gene transfer (HGT) from
an allele of the neomycin phosphotransferase II gene (nptII) with an crops to surrounding soil microorganisms. Our analysis of
internal deletion) with significant sequence similarity to plant trans- antibiotic-resistant bacteria and of the sensitivity of current
genes (e.g., an intact nptII), thus influencing the event through the use techniques for monitoring HGT from transgenic plants to soil
of homologous recombination to boost the detection of transfer17–21.
microorganisms has two major implications for field trial
These studies have been important demonstrations that gene transfer http://www
assessments of transgenic crops: first, HGT from transgenic
occurs, even if HGT was influenced by the methods used to observe it.
plants to microbes could still have an environmental impact
In this article, we use the best measurements of frequencies of gene at a frequency approximately a trillion times lower than the
transfer and the inferred histories of antibiotic-resistant bacteria current risk assessment literature estimates the frequency to
to critique contemporary HGT risk assessments of transgenic crops.
be; and second, current methods of environmental sampling
We show, using the evolution of penicillin-binding protein genes as to capture genes or traits in a recombinant are too insensitive
an example, that experimental limitations preclude measuring for monitoring evolution by HGT. A model for HGT involving
HGT with the sensitivity necessary to dismiss eventual environmental iterative short-patch events explains how HGT can occur at
harm. Therefore, existing data do not justify confidence in the state- high frequencies but be detected at extremely low frequencies.
ments that HGT happens, but at “exceptionally low frequencies3” andthat it is “so rare as to be essentially irrelevant to any realistic assess- Today’s commercial applications of transgenic organisms pose some ment of the risk involved in release experiments involving transgenic of the same types of risk to environment and health as previous appli- plants”22. We offer a different view of the mobile gene ecosystem and a cations, such as the massive release of antibiotics into the environ- model of HGT that we believe is more relevant to assessing environ- ment1,2. When assessing the impact of transgenic organisms, most risk mental risks (Fig. 1).
assessments will consider HGT (gene reproduction and segregation toorganisms or cells separately from the reproduction and segregation of Lessons from Streptococcus pneumoniae
the genome as a whole), ecological lag times and toxicity of the prod- The penicillin-binding proteins (PBPs)—targets of the drug— uct (if it is to be consumed; e.g., see refs. 3,4). These same issues were of Streptococcus pneumoniae with reduced susceptibility to penicillin pertinent to the wide-scale deployment of antibiotics 50 years ago, differ from those of wild-type S. pneumoniae23–25. Loosely speaking, even if they were not fully apparent to those who were assessing the five PBPs contribute to killing and resistance at some concentrations of penicillin (discussed in refs. 26–28). All five PBPs have been The impact of the medical and agricultural use of antibiotics is well changed in some viridans streptococci isolated from the clinic29, understood and described, giving nearly complete retrospective expla- suggesting that, in situ, more than just the two most important PBPs nation for the global spread of antibiotic resistance genes by HGT5–9.
(2b and 2x) might contribute to resistance. Four S. pneumoniae pbp The question of gene transfer is not ‘will it happen?’ but ‘when and genes, through five independent mutations24,30, are reported to change where will it happen?’ A more sophisticated understanding of the way to raise S. pneumoniae’s tolerance of penicillin to 2 µg/ml, the levels genes transfer and ultimately settle into new genomes is required to rec- observed in some clinical isolates25.
oncile divergent claims about the risks of HGT from transgenic crops.
Mosaic genes. Clinical isolates resistant to high levels of penicillin
Descriptions of genomes make clear that HGT has deeply influ- have pbp genes that are mosaics (for an explanation of mosaic genes, enced their structures10–16. Yet attempts to confirm HGT from trans- see Fig. 1) of DNA sequences of pbp genes from at least two (the recip-
genic plants to soil microorganisms in the broader environment have ient and a donor), and possibly more, species24,25. Donor species haveone or more pbp genes that produce proteins with naturally low affini-ties for penicillin. Regions of those genes are found interspersed in the 1New Zealand Institute of Gene Ecology, University of Canterbury, 8020, Private sequences of resistant S. pneumoniae’s pbp genes.
Bag 4800, Christchurch, New Zealand. 2Norwegian Institute of Gene Ecology,POB 6418, N-9294 Science Park, Tromsø, Norway. Correspondence should be The history of mosaic pbp genes in S. pneumoniae illustrates addressed to J.A.H. (email@example.com).
why HGT is profoundly difficult to measure. First, penicillin resistance Published online 31 August 2004; doi:10.1038/nbt1009 was assimilated into the S. pneumoniae genome through successive NATURE BIOTECHNOLOGY VOLUME
Figure 1 Evolution of mosaic alleles by molecular massage. HGT domain
swapping involves moving genes or sub-genes between genomes. Innature, domain swapping extends to significantly different nucleotide sequences. (a) The homology-directed illegitimate recombination model
of Prudhomme et al.63 illustrates how homologous recombination leads
to the insertion of nonhomologous DNA. In this model, insertion of donor DNA (solid boxes) follows the legitimate crossover events of homologousrecombination, with concomitant deletion of intervening sequences ofrecipient DNA (open boxes). Single-stranded DNA corresponding to perhaps a highly divergent allele of a pbp gene, that encodes a PBP with low affinity for penicillin, is taken up by a competent and penicillin- susceptible strain of S. pneumoniae. Short stretches of DNA of near orabsolute identity (≥153 nucleotides, gray boxes) in the otherwise highly divergent donor DNA initiate invasion of the donor strand. Extremely shortstretches of sequence identity (‘microhomology,’ 3–10 nucleotides, gray lines) suffice to define the end of the length of heterologous DNA inserted. (b) The short stretches of highly similar DNA that bring a region
to the threshold of ‘recombination’ are either present by chance or by conditioning. We propose that sequence conditioning begins when biochemical barriers, primarily mismatch repair (MMR)64–67, fail to remove mispaired DNA during recombination (inset). Depending on .com/naturebiotec
the proficiency of mismatch repair (MMR), stretches of DNA fromhomologous (5%–30% divergent) sources may initially be paired, with the invading strand subsequently degraded. MMR can saturate under stress49,68–70 or falter through mutation, allowing some mispairs to escape repair. Stretches of the recipient strand could be massaged into a closer match with the donor DNA over short intervals (black lines in recipient gene). High-frequency HGT could thus leave iterative small http://www
changes that would be mistaken for variation from polymerase errors, if detected at all. This model illustrates the importance of measuring gene transfer frequencies, not just inheritance (transmission) frequencies, for estimating the impact of HGT in the environment.
introductions and replacements of nucleotides sourced from highly strains with much higher efficiency (by homologous recombination), diverged donors (Fig. 1b). Gene transfers between species most fre-
as could combinations of recombinant genes assembled in one or quently result in short stretches of recombination, the mosaicism more different strains38. The speed at which penicillin resistance observed in pbp genes, which are invisible to most analyses (see chapter spread by subsequent gene transfer events could have accelerated by J.A.H in ref. 31). Second, the lag32 between environmental impact exponentially from the point in time at which the alleles were first and genesis of the recombinant phenotype is an unpredictable variable.
assembled, far exceeding the speed at which emergent clonal lineages Although it took 50 years for high-level penicillin-resistant S. pneumo- reproduced or colonized new environments.
niae to become 21.5% of the isolates in the United States33, that out-come could not have been predicted in 1950 anymore than in 2004.
Implications for monitoring
Ecological lag time. The time taken for a trait to emerge is partly a
The purpose of a transgenic crop field trial designed to assess HGT function of the adaptive value of a new gene, but the strength of selec- is to produce meaningful measures of potential harms arising from tion or absence of selection cannot always be known in advance34,35, gene transfer and estimate the safety margins needed to avoid them.
and any adaptive value must overcome the inhibiting effect of the A verified trial would, either through scale or other design features, dominant flora36. In some cases, the emergent phenotype may be seen produce outcomes that are both qualitatively comparable to the range, only when the environment changes, or the microbe changes environ- and proportional to the magnitude, of impacts that could be expected ments, as in the evolution of antibiotic resistance before selection from full releases.
(e.g., see reviews from J.A.H. group8,37). Only recently have formal Detection limits. Could past trials have detected HGT at a fre-
experiments attempted to begin measuring the influence of selection quency below which any environmental harm would arise? Techniques being used to monitor HGT in soil have sampling limits of about one HGT introduces another complexity in attempts to measure the recombinant bacterium in 108–1011, and these experiments uniformly lag time. When genes evolve by transfer rather than through organis- yield no detectable recombinants unless special conditions are mal reproduction, neither the generation time nor the geographical applied4,17–21,39–41. Some authors have imposed additional assump- range of the organism necessarily limits the lag time. This last point is tions about barriers to HGT and extrapolated an estimated frequency particularly relevant to attempts to measure HGT in field trials: the of many orders of magnitude less than their sampling limits, that is, to combinatorial development of mosaic genes in decade time scales fol- less than one event in 1016–1017 (refs. 22,39). Trials verified as relevant lows from the flow of genes across the globe, not through the genera- to a risk assessment would therefore have features that permitted tion of variation within plots. In the case of S. pneumoniae, once one detection of recombinants at HGT frequencies <10–17. Clearly, no low-affinity pbp allele was made, it could be transferred between published trials have been that powerful.
VOLUME 22 NUMBER 9 SEPTEMBER 2004 NATURE BIOTECHNOLOGY
Figure 2 Implicit assumptions in HGT monitoring. The search for
recombinant microorganisms that could arise in the soil surroundingtransgenic plants invariably incorporates a step where the bacteria areisolated and cultured and those displaying a phenotype that can beselected (e.g., antibiotic resistance) or screened (e.g., PCR or intensity of fluorescence54) are taken as recombinants. Every selection/screen has a threshold monitoring range (dotted horizontal lines perpendicular to the y-axis). Recombinants that do not display in this range will not be detected. For example, an investigator-imposed threshold penicillinconcentration would miss recombinant strains of S. pneumoniae that are resistant to other levels of penicillin but may arise at much higherfrequencies and would lead to false confidence that the use of penicillinat such concentrations would be of low risk to the evolution of clinically important penicillin-resistant strains. The challenge for future monitoringproposals is to justify that the monitoring range is within the reach of the population at the temporal and population scales being tested.
Even if they had been, would this detection limit verify the trial? 5 × 1030, with an average turnover of three years43. Gene transfer mag- Existing knowledge of S. pneumoniae pbp genes can be used to answer nitudes are conservatively 100 times this already impressive scale .com/naturebiotec
this question. Majewski et al.42 recovered single-gene S. pneumoniae because each organism may host, over its lifetime, 10–100 horizontally recombinants at a frequency of approximately 10–6 using DNA from mobile elements (e.g., viruses or conjugative plasmids). These approx- donor sources that have diverged by 17%–18% in DNA sequence. The imations are also consistent with estimates of gene transfer derived degree of sequence divergence between donor and recipient pbp alleles from observations of the viral load in the world’s oceans (see chapter used in this study was in the middle of the range seen in alleles actually donated to penicillin-susceptible clinical isolates of S. pneumoniae The number of transgene transfers to soil microbes thus can be esti- http://www
(14%–25%25,30). Using this transmission frequency as a guide, the pre- mated based on derived HGT frequencies and the size of the microbial dicted frequency of S. pneumoniae with one recombination event per population (Table 1). For example, a transmission frequency of 10–12
pbp gene is 1 × 10–24 [(1 × 10–6)4]. (This estimate would be valid even if could result in 4,000 recombinants per square meter of top soil (based only PBP2b and 2x had to change because a minimum of two changes on 5 × 1028 bacteria per 1.4 × 1013 m2 of top soil43). Ten recombinants in each of PBP2b and 2x are thought to be required24.) For strains with could be expected in 250 m2 if the gene transmission frequency were six events (e.g., possibly the South African isolate described in refs.
10–17, with upwards of a trillion recombinants among the nearly 25,27), the frequency would be 1 × 10–36 [(1 × 10–6)6]. In theory, the 70 million hectares of transgenic crops44. Were HGT truly rare, on the evolution of penicillin resistance and its consequences has resulted order of the inverse of Avogadro’s number (10–24), 5,000 recombinants from events predicted to be 107–1019 times rarer than frequencies of would be expected in the estimated 11.4 million hectares currently HGT estimated to be occurring in soil.
planted in Bacillus thuringiensis (Bt) corn45. Although it might seem Whereas the low-affinity alleles of all the different mosaic pbp genes that these numbers should be big enough for trials to detect recombi- in a given strain of S. pneumoniae were probably not built into their nants, distributed among the normal flora a minimum of 4 × 108 m3 final complexity at each locus from one lucky scoop out of the pool of (500 million metric tons) of soil would have to be sampled to find one DNA surrounding them, contemporary experiments on transgenic (this estimate is based on 5 × 1027 bacteria per 1.14 × 107 hectares of organisms impose that requirement on the organisms being moni- top soil; see Table 1).
tored in a relatively small area for comparatively short times, and relyon a high frequency event to overcome unpredictable lag times so that Implications for risk assessment
at most only a trillion culturable organisms would be needed to reveal When HGT is considered in relation to risk assessments, we must con- HGT. Gene transfers that result in intermediate phenotypes or pheno- sider not only whether HGT is occurring, but also the critical issue of types different from those expected by the investigator are lost (Fig. 2).
its consequences for health and environmental safety. The latter is A verified trial for studying HGT, therefore, would require a protocol to screen approxi-mately 1025–1037 bacteria for penicillin resist- Table 1 Estimated number of HGT events from transgenic plants to soil microbes on
the basis of derived HGT frequencies and microbial population size
cultured and plated at densities of 1010/Petridish, a minimum of 1015 and 1027 Petri dishes, respectively (and many times more if other culturable bacteria from the environ- Recombinants in Bt corn fields (global)b required to detect one recombinant arising de Size of soil sample for one recombinanta,b,c 3 × 105 T 3 × 1010 Td
3 × 1012 T
Ecological issues. A new respect for the
scale of the microbial world is required to aBoldface text for frequencies indicates frequencies higher than any reports of environmental HGT that we are aware of appreciate the detection problem. In some (unless special recipients were used); boldface text for sample sizes indicates sample sizes larger than those in any studiesthat we know of that have examined the full genomic content of the sample. bBased on the following calculation: 5 × 1027 soils, like rich top soils, there are approxima- bacteria/5,000 recombinant bacteria) × (g soil/2 × 109 bacteria) × (m3/1.3 × 106 g soil). cg, grams; kg, kilograms; T, metric tely two billion microorganisms per gram43.
tons. dThis amount of soil would fill a train of 500 million (standard 70 US tons) boxcars, long enough to encircle the equa-tor 192 times. eAssuming 1% of soil microorganisms are culturable71,72.
NATURE BIOTECHNOLOGY VOLUME
dependent on the likely impact of the newly acquired trait in its eco- applications that introduce HGT risks and adjust the pace of their logical and geographical context. In the case of transgenic plants release to match developments in our ability to monitor at relevant expressing Bt toxins, for example, the ubiquity of B. thuringiensis in sensitivities, recognizing that the technology of safety monitoring soil was considered by the US Environmental Protection Agency lags behind the technology of genetic engineering. The slowdown may(EPA; Washington, DC, USA) to reduce the environmental impact of recombinant Bt toxin transgenes, even if they did transfer to soil From a technological standpoint, some groups are already develop- microorganisms41. However, it is useful to invoke what is known about ing vectors carrying genes that are expressed in a larger number of S. pneumoniae for evaluating the EPA assessment.
species, including those that cannot be cultured, thus extending our Bt is a shorthand for the cry toxin genes, modified from those first understanding of gene movement in the context of the larger soil bio- isolated from the soil bacterium B. thuringiensis, that confer resistance diversity54. Even more intriguing are developments that map the to various insect pests of plants. The cry genes appear to be of mosaic mobile gene landscape and thus develop indirect measures of HGT construction, like the pbp genes46. The combinations of domains activity and gene diversity in particular places and times55–57. As onedistributed among the various cry genes alter the range of species of us (J.A.H) has previously noted58, these approaches look promisingthat find the protein toxic. In this regard, it is noteworthy that because they capture the novel gene diversity predicted to emerge fromB. thuringiensis has “a significant history of mammalian pathogenic- HGT. The detection limits of these new developments are still not ity”46 and is thus not irrelevant to food safety or other environmental known, and will probably fall short of detecting the early bouts of issues. Before human application of penicillin, the EPA might have short-patch interspecies recombination (Fig. 1b), but their innovation
similarly dismissed concern about the trafficking of the extremely removes the need to find a particular DNA sequence within a sea of .com/naturebiotec
small PBP protein domains because pbp genes are ubiquitous in DNA, the very factor that limits conventional approaches.
human flora (low-affinity alleles originate in normal human commen- HGT binds the microbial community into a complex network59,60 sals, such as the viridans streptococci). Moreover, large fragments of even at intuitively ‘low’ frequencies of transfer (≤10–17), allowing alle- the modified forms of cry transgenes, which may not be identical to les of genes to evolve on a global scale7,30,61. HGT has been dismissed the gene found in the soil bacteria47, persist through digestion in pigs by commentators in response to concerns about the possible impact of and exit with feces48. DNA from cry genes was detected only when transgene migration from released transgenic crops, even though the http://www
their source was Bt corn, suggesting that normal soil organisms are less scale of commercial production already makes such risks plausible.
likely to contribute to any recycling through animals, making the Gene transfer is facilitated by many different kinds of vectors and envi- transgene DNA relatively more available to gut and soil bacteria.
ronmental conditions and is not restricted to microbes. Among a Mosaic genes are becoming the norm in antibiotic-resistant micro- number of different and plausible transgene vectors are viruses capa- bial flora49. For example, three genes (parC, parE and gyrA) must ble of crossing even the plant-animal divide62. The variety of transfer change in S. pneumoniae for it to become resistant to clinical levels of paths and vectors, and the number of genomes that could serve as tem- the drug ciprofloxacin. Resistant strains carry mosaic alleles of each of porary or permanent homes for transgenes (or parts thereof), make these genes, with the viridans streptococci again being the most likely the speculative calculations presented here highly conservative.
donors50. The mosaic regions vary from 0.6%–12% in DNA sequence Contrary to the conclusion of others3, we believe that new approaches from susceptible strains, and some resistant strains have eight putative to monitoring environmental scale applications of transgenic organ- interspecies domains distributed over the three genes50. Domain swapping can be a powerful route to protein diversity that is revealed only by the introduction of new selective pressures and niches, not ACKNOWLEDGMENTS
We thank C. Amábile-Cuevas, H. Cochrane, D. Bean and R. Mann for critical
always predictable from known biochemical function and apparent comments on the manuscript. J.A.H. acknowledges support from the Marsden Fund of New Zealand (M1042) and the Brian Mason Trust.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
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NATURE BIOTECHNOLOGY VOLUME
DENKA to strengthen Capital Alliance with SEI Corporation in the Field of Lithium-ion Secondary Battery Technology DENKA (president: Shinsuke Yoshitaka) and SEI Corporation (headquarters: Tsu, Mie; representative director: Takehiko Sawai; hereafter “SEI”) have reached an agreement to accelerate the development of electrode material technologies, with a focus on high conductivity (lo