Problems 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
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
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
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-
(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
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
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. Concluding remarks COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
The consequences, if not the precise frequencies, of HGT in microor-
Published online at http://www.nature.com/naturebiotechnology/
ganisms are becoming well known to those who suffer from bacterialinfections that resist treatment by the least toxic and least expensive
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