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Horizontal ‘gene drives’ harness indigenous bacteria for bioremediation

19 Nov 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 735886
TL;DR: Pilot experiments show that vectors only persist in indigenous populations when “useful,” disappearing when this carbon source is removed, which could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.
Abstract: Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and environmental concerns. Here, we develop a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in E.coli led to degradation rates of 60-99% of target hydrocarbon substrates. Mating experiments, fluorescence microscopy and TEM revealed indigenous bacteria could obtain these vectors from E.coli through several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic exchange through nanotubes. Inoculating petroleum-polluted sediments with E.coli carrying the vector pSF-OXB15-p450camfusion showed that the E.coli die after five days but a variety of bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that vectors only persist in indigenous populations when “useful,” disappearing when this carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading pollutants while providing minimal ecosystem disturbance.

Summary (4 min read)

1 A Composition and a Commodity

  • Historians of science are obviously expected to study many different aspects of any book as instructive and influential as the Origin.
  • 3 Analogia, a Fortiori and Vera Causa Darwin’s case for the adequacy, the competence of natural selection as a cause of species origins in branching adaptive descents invokes an analogy with the form of a traditional analogy as proportionality: the struggle for existence is to wild animals and plants as a human breeder is to domestic ones.

4 The First Four Chapters (I–IV)

  • Introducing the first (1859) edition of the Origin requires trawling through its chapters— after a warning and a suggestion: Darwin’s prose is often not very lucid; for Darwinism expounded more clearly one goes to the books of Alfred Russel Wallace.
  • By contrast, the causes of selection under domestication and in nature—the human breeder’s practices and the struggle for life—are entirely unalike, but their selective consequences for survival and reproduction are the same in kind though not in degree.
  • The principal causal theme of the fourth chapter (IV), on natural selection, is anticipated here when this struggle is cited as the cause accumulating hereditary variation selectively and so adaptively.
  • In the later cluster of four chapters (X-XIII), Darwin will find no explanatory work for sexual selection.

5 Three General Evidential Considerations

  • Writing on the 22nd of May 1863 to George Bentham, the eminent botanist and author when young of a book on logic, Darwin insisted that: the belief in natural selection must at present be grounded entirely on general considerations.
  • (3) & chiefly from this view connecting under an intelligible point of view a host of facts.
  • The second general consideration is the analogical comparing and contrasting of nature’s selection with man’s in establishing what nature’s selection can do in its much longer run.
  • So much then for the Origin’s first four chapters.

6 On the Middle Five Chapters (V–IX)

  • Darwin’s three general evidential considerations do not map onto the Origin’s three clusters of chapters.
  • In fact he has talked only once or twice in passing of chance variations, and the larger aim of the chapter is not limited to correcting any misleading impressions such talk might have given.
  • For what the chapter mainly argues for is a unification thesis.
  • Variations in domestic and wild plants and animals all conform to the same laws.
  • After this chapter on the laws of variation comes a chapter (VI) often anticipating and countering various reasons for thinking natural selection incapable of producing new species from old, because some species have features that selection can not produce, especially such organs of extreme complexity and perfection as the eye.

7 On Those Later Four Chapters Preceding the Last One (X–XIII)

  • These later four chapters take up three clusters of topics; for the first (X) is on geology— which, recall, has just had the last of the middle five devoted to it—the next two (XI, XII) to biogeography and the fourth (XIII) to taxonomy and morphology including comparative embryology.
  • It is worth dwelling on these themes as Darwin gives no clue as to why he has treated these three clusters of topics in the order he adopts.
  • Naturally, Darwin explains this close likeness as due to descent, and declares descent supported evidentially by its yielding this explanation; but he does so without explicitly complementing this declaration by urging that the slight differences between the extant and extinct species are best explained as due to selection.
  • A second reason for that exegetical mistake being useful concerns alternative explanatory options.
  • Other resemblances might be explained as due to common fittings, adaptations, providential or otherwise, to common ways of life, aquatic life, say.

8 Understanding the Divide

  • This task of understanding the relations between the opening four chapters and these later four involves difficult issues of interpretation; and they are not issues clarified in Darwin’s closing chapter.
  • Darwin’s extrapolationary theorising is not then from ontogeny to phylogeny as ontogeny writ large, but from intraspecific adaptive divergence—as ethnologically, geologically, geographically and ecologically interpreted—to interspecific adaptive diversification.
  • Von Baer had insisted on distinguishing between the level of organisation and the type of organisation; and Darwin’s account of progress in branching descents conforms to that distinction.
  • In many fields of inquiry in Darwin’s day, as before and since, theorists saw themselves as having to adjudicate between historical, functional and structural considerations.
  • So, the divide between the early four chapters (I-IV) and those four later ones (X-XIII) is indeed a divide between what provides and what requires explanation, between what has explanatory priority and what does not.

9 Innovation and Conservation

  • Historians are trained and paid to be sceptical about hyperbolic declarations that some famous book contained a totally new way of thinking that changed everything instantly for everyone.
  • They are also inclined to be pluralists, and so sceptical of claims that some famous author was driven in his or her whole life and work by some one vision, ambition or allegiance.
  • Start with the heavens and the solar system.
  • There were precedents for not taking up these topics, precedents in the writings of two authors who were consciously allied on such issues, and whose authority Darwin took very seriously: Herschel (John, not to be confused with his nebular father William) and Lyell.
  • On species origins themselves, Darwin broke not just with Lyell’s commitment to independent, special creations of fixed species, but also with his commitment to the providential determination of the timing and placing of those events by adaptive considerations alone.

10 Species: Explaining and Explaining Away

  • On species themselves, it is natural to presume that the long argument of the Origin required a radically new conception of what species are.
  • For the distinguishing of one species from another would be arbitrarily settled by naturalists’ naming decisions rather than by nature’s real distinctions; and that any general doctrine, as to what plant or animal forms are to be ranked and named as species rather than mere varieties, would likewise be conventional not natural.
  • To that extent Darwin was far from dismissing or ignoring these diagnostic criteria, but was taking them all, insofar as they were consistent with gradual transmutations, as determining his explanatory challenge.
  • In his thirteenth chapter, Darwin did not say that common descent and natural selection made worthless all existing classificatory work, also known as Similarly with classification generally.

11 Alignments

  • Even a glance at what the Origin rejected and what it retained of earlier theories and practices shows that the book was not an attempt to replace all that went before.
  • What comparisons and contrasts between Darwin and Wallace can do for any understanding of the Origin, is to show that the book was not presenting an obvious next step in the advancement of public biological science; and, equally, that it was not presenting a purely personal expression of one man’s desire for truth.
  • By the time one gets to Boyle and his friend Newton, the Dewey–Mayr historiography is paying a big a price in credibility for its ignoring of Greek, especially Epicurean, alternatives to Plato and Aristotle; and the credibility is never recoverable as one turns to the mid-eighteenth century.
  • Cartesian or Newtonian matter, like Platonic and Aristotelean form, is then no longer an inherent, requisite grounding for any botanist’s, zoologist’s or geologist’s commitment to the lawfulness of nature.
  • All the developments associated with industrialisation and urbanisation in the industrial revolution decades were consequently far from displacing the prior prominence of agrarian, financial and imperial capitalisms, and for at least four reasons.

15 Concluding Remarks About Historians, Philosophers and Scientists

  • Exceptionless generalisations about historians of science are no more to be expected than universal truths about historians of politics or of art; but there are some commonplaces that many historians of science accept.
  • Staying with this threefold contrast between historians, scientists and philosophers, let me address a limitation in what I have written in this essay.
  • Some historians of science fortify themselves against such forgetfulness by formulating their interpretations of Darwin’s aims and intentions using only terms, concepts and categories that Darwin himself could have used in his time and place.
  • A commonplace says that the authors need to study the past in order to better understand the present; and that this commonplace applies to the past and present of science no less than to the past and present of economics or of war and peace.

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www.nature.com/scientificreports
Horizontal ‘gene drives’
harness indigenous bacteria
for bioremediation
Katherine E. French
1*
, Zhongrui Zhou
2
& Norman Terry
1
Engineering bacteria to clean-up oil spills is rapidly advancing but faces regulatory hurdles and
environmental concerns. Here, we develop a new technology to harness indigenous soil microbial
communities for bioremediation by ooding local populations with catabolic genes for petroleum
hydrocarbon degradation. Overexpressing three enzymes (almA, xylE, p450cam) in Escherichia coli
led to degradation of 60–99% of target hydrocarbon substrates. Mating experiments, uorescence
microscopy and TEM revealed indigenous bacteria could obtain these vectors from E. coli through
several mechanisms of horizontal gene transfer (HGT), including conjugation and cytoplasmic
exchange through nanotubes. Inoculating petroleum-polluted sediments with E. coli carrying the
vector pSF-OXB15-p450camfusion showed that the E. coli cells died after ve days but a variety of
bacteria received and carried the vector for over 60 days after inoculation. Within 60 days, the total
petroleum hydrocarbon content of the polluted soil was reduced by 46%. Pilot experiments show that
vectors only persist in indigenous populations when under selection pressure, disappearing when this
carbon source is removed. This approach to remediation could prime indigenous bacteria for degrading
pollutants while providing minimal ecosystem disturbance.
Oil spills in recent decades have le a long-term mark on the environment, ecosystem functioning, and human
health
13
. In the Niger Delta alone, the roughly 12,000 spills since the 1970s have le wells contaminated with ben-
zene levels 1,000× greater than the safe limit established by the World Health organization and have irreparably
damaged native mangrove ecosystems
4,5
. Continued economic reliance on crude oil and legislation supporting
the oil industry mean that the threat of spills is unlikely to go away in the near future
6
.
At present, there are few solutions to cleaning up oil spills. Current approaches to removing crude oil from
the environment include chemical oxidation, soil removal, soil capping, incineration, and oil skimming (in
marine contexts)
7,8
. While potentially a ‘quick x,’ none of these solutions are ideal. Soil removal can be costly
and simply moves toxic waste from one site to another
9
. Chemical oxidants can alter soil microbial community
composition and pollute groundwater
10
. Incineration can increase the level of pollutants and carbon dioxide in
the air and adversely aect human health
11
. Practices such as skimming only remove the surface fraction of the
oil while the water-soluble portion cannot be recovered, negatively aecting marine ecosystems
12,13
.
Synthetic biology has now given us the tools to tackle grand environmental challenges like industrial pollu-
tion and could usher in a new era of ecological engineering based on the coupling of synthetic organisms with
natural ecosystem processes
1418
. Consequently, using bacteria specially engineered to degrade petroleum could
present a viable solution to cleaning up oil spills in the near future. Previous studies have identied which bacte-
rial enzymes are involved in petroleum hydrocarbon degradation (reviewed in references
9,19
) and have engineered
bacterial enzymes like p450cam for optimal invivo and invitro degradation of single-substrate hydrocarbons
under lab conditions
20,21
. However, there are several critical gaps in or knowledge of engineering bacteria for
oil-spill bioremediation. First, we know little about how the performance of these enzymes compare and which
enzyme would present an ideal target for over-expression in engineered organisms. Second, it is unclear how
well engineered organisms can degrade petroleum hydrocarbons compared to native wild-type bacteria which
naturally degrade alkanes, such as Pseudomonas putida. ird, the environmental eects of engineered bacteria
on native soil populations are unclear. For example, do these bacteria persist over time in contaminated soils?
OPEN

USA.

*
email: katherine.french@
berkeley.edu

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Although the use of genetically modied bacteria in bioremediation is attractive, this solution faces signicant
regulatory hurdles which prohibit the release of genetically modied organisms in the environment
22
.
Here, we propose a new bioremediation strategy which combines synthetic biology and microbial ecology and
harnesses natural processes of horizontal gene transfer in soil ecosystems. We screened ve enzymes involved
in petroleum degradation in E. coli DH5α (alkB, almA, xylE, ndo and p450cam) to identify (1) where these
enzymes localize and their eect on crude oil using advanced microscopy and (2) to asses each enzymes ability
to degrade three petroleum hydrocarbon substrates (crude oil, dodecane, and benzo(a)pyrene) compared to two
wild type bacteria (Pseudomonas putida and Cupriavidus sp. OPK) using bioassays and SPME GC/MS. Based on
these results, we selected one vector (pSF-OXB15-p450camfusion) to determine whether small, synthetic vectors
carrying catabolic genes could be transferred to indigenous bacteria found in petroleum-polluted sediments and
whether this shi in community metabolism could increase rates of pollutant degradation.
Results and discussion
Overexpression of petroleum hydrocarbon-degrading enzymes in E. coli. To compare the locali-
zation and activity of known petroleum hydrocarbon-degrading enzymes, we inserted ve enzymes (alkB, almA,
xylE, ndo, and p450cam) and required electron donors into the vector backbone pSF-OXB15 using Gibson
Assembly
23
(SI Fig.1). To identify where each enzyme localized within E. coli DH5α, we tagged each enzyme
with a uorophore (gfp or mcherry). Fluorescence microscopy revealed that alkB was localized to bacterial
cell membranes and almA was found throughout the cytoplasm. e camphor-5-monooxygenase camC from
the p450cam operon was expressed throughout the cell cytoplasm while another enzyme in the operon, the
5-exo-hydroxycamphor dehydrogenase camD, was expressed within a small compartment at one end of the cell
(Fig.1A). e dioxygenase ndoC from the ndo operon was also localized to a small compartment at one end of
the cell. e dioxygenase xylE was found in small amounts in the bacterial cell membrane and larger amounts in
a microcompartment at one end of the cell. In all cases, these compartments were 115–130nm wide and could
be seen in young, mature and dividing cells. e presence of microcompartments in E. coli expressing p450cam,
ndo, and xylE could reect the known use of protein-based microcompartments by bacteria to concentrate
highly reactive metabolic processes
24
.
Over-expression of all ve enzymes imbued E. coli DH5α with metabolism-dependent chemotactic behavior,
where cell movement is driven towards substrates aecting cellular energy levels
25
. E. coli DH5α do not have
agella, but rather, moved towards petroleum hydrocarbon substrates via twitching
2628
. Fluorescence microscopy
showed E. coli DH5α expressing alkB ‘clinging’ to oil droplets (Fig.1B) and those expressing xylE seemed to use
the compartment-bound enzyme as a ‘guide’ towards crude oil (SI Fig.2). Both behaviors mimic the interactions
of wild-type oil-degrading bacteria
7
.
Fluorescence microscopy also revealed for the rst time the key role of extracellular enzymes in degradation of
petroleum hydrocarbons. ree enzymes, alkB, almA, and p450cam were found in extracellular vesicles ranging
in size from 0.68μm to 1.67μm (SI Figs.3 and 4). ese vesicles were only seen when E. coli DH5α was exposed
to petroleum hydrocarbons. ey are larger than minicells (which range from 200–400nm in diameter)
29
and
seem to serve some other function. Confocal images suggest that these vesicles may come into contact with oil
droplets, potentially attaching to (or merging with) their surface (SI Fig.4).
We also found three enzymes, alkB, xylE, and p450cam, within the E. coli exopolysaccharide (EPS) matrix.
AlkB and xylE were concentrated around the 500nm pores within the EPS and found dispersed in smaller
amounts throughout the exopolysaccharide (Fig.1C; SI Fig.5). In contrast, p450cam was distributed in high
amounts throughout the EPS. Cryotome sectioning of the EPS from bacteria expressing p450cam indicates that
the monooxygenase camA from the p450cam operon co-localized with a second enzyme involved in hydro-
carbon degradation, the dehydrogenase camD (SI Fig.6). Protein levels of EPS varied signicantly among the
dierent strains of wild-type and engineered bacteria (ANOVA test, F
7,16
= 11.3, p < 0.001, adjusted R
2
= 0.76).
Figure1. Expression and localization of bacterial monooxygenases and dioxygenases involved in petroleum
degradation in E. coli DH5α. (A) Structured Illumination Microscopy (SIM) image of E. coli DH5α expressing
proteins involved in petroleum degradation (cam A, B, C and D) from the CAM plasmid in E. coli. camC(fused
to mcherry) was found throughout the cell while camD(fused to gfp) localized to a microcompartment at one
end of the cell. e scale bar is 5μm. (B) E. coli DH5α expressing alkB fuse to gfp were found clinging to spheres
containing crude oil, mimicking a behavior seen in wild-type oil-degrading bacteria. (C) EPS from E. coli DH5α
expressing xylE. Gfp-tagged xylE were found around small pores (ca. 500nm) within the EPS matrix. (B) and
(C) were taken using the GFP lter on a Zeiss AxioImager M1.

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Bacteria expressing alkB (0.84 ± 0.04mg/ml), xylE (0.85 ± 0.13mg/ml), and p450cam (0.97 ± 0.06mg/ml) had
higher levels of EPS protein than E. coli DH5α expressing the empty vector pSF-OXB15 (0.44 ± 0.02mg/ml) (SI
Fig.77 and were comparable to the EPS protein levels of Cupriavidus sp. OPK (1.2 ± 0.15mg/ml), a bacteria
known to use biolms to degrade crude oil
7
. Although previous studies suggest that EPS may be involved in the
extra-cellular metabolism of environmental pollutants
7,3034
, this is the rst study to identify several enzymes
which may play a role in this process.
Finally, extracellular enzyme expression also inuenced the size of the oil droplets within the cell culture
media. For example, E. coli DH5α expressing xylE produced very small oil droplets (primarily > 1μm in diam-
eter) of crude oil while those expressing alkB and almA produced droplets ranging in size from 1μm-120μm in
diameter (SI Fig.8). Although xylE was not seen in vesicles, confocal suggests that crude oil droplets can ow
through pores in biolms and may become coated in EPS in the process. Potentially, attachment of enzymes to
oil droplets (through fusion with vesicles or contact with EPS) may inuence how fast a droplet is degraded over
time. Previous studies have shown that vesicles embedded with enzymes can catalyze chemical reactions
35,36
. In
addition, Dmitriev etal. have shown that two bacteria, P. putida BS3701 and Rhodococcus sp. S67, use vesicular
structures containing oxidative enzymes which attach to and play a role in degradation of crude oil droplets
37
.
Our results thus suggest that bacterial monooxygenases and dioxygenases involved in petroleum hydrocarbon
degradation may be involved in multiple, complex inter and intra-cellular processes that lead to the degradation
of crude oil.
Comparison of enzyme activity. To determine which enzymes were most useful for degrading long-
chain hydrocarbons, polyaromatic hydrocarbons (PAHs), and crude oil, we conducted 96-well plate assays
exposing wild-type and genetically engineered bacteria to 1% (v/v) of dodecane, benzo(a)pyrene or crude oil.
We found that bacteria engineered to over-express specic enzymes in petroleum degradation were able to
degrade single-carbon substrates better than the wild-type bacteria P. putida and Cupriavidus sp. OPK. One
way analysis of variance (ANOVA) of the assay data showed that there was signicant variation in bacterial
growth when exposed to dodecane (F
8,31
= 33.4, p = < 0.001), benzo(a) pyrene (F
8,31
= 73.03, p = < 0.001) and
crude oil (F
8,31
= 240.6, p = < 0.001). When exposed to dodecane, E. coli DH5α expressing p450cam increased
in biomass the most (139.4%), followed by E. coli DH5α expressing xylE (136.3%), alkB (120.8%), and almA
(97.6%) (Fig.2A). Expressing p450cam and xylE led to signicantly greater conversion of dodecane to biomass
compared to P. putida (t = 4.71, df = 3.17, p < 0.01 and t = 4.41, df = 3.17, p < 0.01 respectively). Solid-Phase Micro-
Extraction (SPME) GC/MS analysis of these cultures revealed that all three bacteria degraded 99% of dodecane
in 10days. When exposed to benzo(a)pyrene, P. putida had the greatest increase in biomass (119.2%) followed
by E. coli DH5α expressing almA (117.1%), xylE (94.8%), and p450cam (90.8%) (Fig.2B). T-tests showed there
was no signicant dierence in the biomass of P. putida and these three strains (p > 0.10). SPME GC/MS showed
that E. coli expressing P450cam, almA and xylE degraded 90%, 97% and 98% of the benzo(a)pyrene respectively
while P. putida degraded 86%.
In contrast, when engineered and wild-type bacteria were exposed to crude oil, P. putida converted the oil to
biomass more eciently, increasing in biomass by 110.9% (Fig.2C). Only two genetically engineered bacteria,
E. coli DH5α expressing p450cam and almA, had comparable increases in biomass to Cupriavidus sp. OPK
(61.93%, 52%, and 48.7% respectively). e assay was repeated with crude oil stained with Nile Red and rates of
degradation were determined according to French and Terry
7
. P. putida degraded 79% of crude oil while E. coli
Figure2. Growth of wild-type and engineered bacteria on dodecane (A), benzo(a)pyrene (B), and crude oil
(C). Wild type strains are denoted as ‘Cupriavidus’ and ‘P. putida.’ Synthetic strains are denoted according to
what enzyme they are engineered to express (e.g. alkB, almA). e two negative controls are a control with the
carbon substrate but no cells and E. coli DH5α transformed with the vector backbone used in the experiment
(pSFOXB15) but without genes inserted for hydrocarbon degradation. Optical density measurements were
taken at OD 600nm. Scale bars show standard error of the mean.

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DH5α expressing p450cam and almA degraded 64% and 60% respectively. E. coli expressing alkB, xylE, and ndo
only grew ~ 25% and degraded 35–40% of crude oil. e high performance of p450cam when exposed to crude
oil likely reects the enzymes known substrate promiscuity
38,39
which makes it a better catalyst for degrading
crude oil, a complex substrate made of over 1,000 compounds
40
.
Vector-exchange between E. coli DH5α and indigenous bacteria. To determine whether our
engineered bacteria could transfer non-conjugative, synthetic vectors containing petroleum-degrading genes
to indigenous soil and marine bacteria, we conducted a series of mating experiments. We found that wild-type
bacteria readily received the vector pSF-OXB15-p450camfusion through horizontal gene transfer (HGT) (Fig.3;
SI Fig.9). Frequencies of transformation ranged from 19 to 84% in 48h depending on the recipient species (SI
Table1) and were > 90% aer seven days of incubation for all tested species. Plasmid expression was stable for
over three months in the absence of antibiotic pressure. Although our vectors carried a ColE1 origin of replica-
tion, this did not seem to present a barrier to HGT. is agrees with previous studies which suggest ColE1 plas-
mids can be found in wild bacteria
41
and wild-type bacteria can receive ColE1 plasmids from E. coli
42
.
HGT can occur through transformation, transduction, conjugation, transposable elements, and the fusing
of outer membrane vesicles (OMVs) from one species to another
43,44
. To test whether wild-type bacteria could
take up naked plasmids from cell culture, we adding 1μl of puried plasmid (at a concentration of 1ng/μl and
10ng/μl) to LB cultures containing wild-type bacteria and by spreading diluted vectors onto agar plates. We saw
no transformed cells. In addition, neither uorescence microscopy or TEM showed OMV production or release
by the E. coli DH5α strains created in this study. OMVs are 50–250nm in diameter
45
, much smaller than any of
the vesicles produced by our strains.
To determine whether HGT of the synthetic vectors to wild-type bacteria was achieved through mating,
we conducted TEM of wild-type bacteria aer exposure to E. coli DH5α carrying pSF-OXB15-p450camfusion.
TEM suggests several mechanisms of HGT through direct cell-to-cell contact may explain how vectors were
transferred between transgenic E. coli DH5α and wild-type bacteria
46
. In our study, we found E. coli DH5α and
wild-type bacteria engaging in DNA transfer through conjugation and cell merging and in cytoplasmic transfer
via nanotube networks. TEM showed E. coli DH5α expressing p450cam tethered to wild-type cells by conjuga-
tive pili over long distances (Fig.4A), the formation of mating pair bridges between wild-type cells and E. coli
DH5α (Fig.4B), E. coli with conjugative pili (Fig.4C), and E. coli and wild-type cells connected via nanotubes
(Fig.4D) (see also SI Fig.10). Although the plasmids used in this study were non-conjugative, such plasmids
can be mobilized and transmitted via conjugation in the presence of other conjugative plasmids or by merging
with conjugative plasmids
4749
. Previous studies have also shown that plasmids (and chromosomal DNA) can be
transferred through cell-contact dependent transfer without the use of conjugative pilii. is mechanism was rst
observed in 1968 in Bacillus subtilis and subsequently in other species (e.g. Vibrio, Pseudomonas, Escherichia)
(reviewed in
49
). For example, Paul etal.
50
found that lab strains of E. coli could transfer plasmid DNA to Vibrio
through this process. Nonconjugal plasmids can also be transferred from between bacteria through nanotubes
51
.
Dubey and Ben-Yehuda
52
show in their classic paper that gfp molecules, calcein, and plasmids could be trans-
ferred between B. subtilis cells. ey also show that a non-integrative vector carrying a resistance marker from
B. subtilis could be transferred to Staphylococcus aureus and E. coli (with recipient cells expressing antibiotic
resistance). is transfer was rapid and could happen in as little as 30min. In our study, it is impossible to say
denitively by which mechanism our vectors were transferred from E. coli DH5α to the wild-type bacteria and
in reality multiple mechanisms of transfer may be possible.
We conducted an additional experiment to determine the survival rate of engineered bacteria in petroleum
polluted sediment from a former Shell Oil renery in Bay Point, CA and whether these genes could be transferred
to native, complex soil microbial communities. is sediment is contaminated with high levels of petroleum
hydrocarbons, arsenic, heavy metals, and carbon black. At D
0
, E. coli DH5α containing the plasmid pSF-OXB15-
p450camfusion were seen in aliquots of contaminated sediment and there were no autouorescent bacteria
Figure3. Expression of pSF-OXB15-p450camfusion in the marine bacteria Planococcus citreus. (A) DIC image
of wild-type P. citreus (no exposure to E. coli). ese bacteria are coccoid-shaped with cells found individually or
in groups of 1–4. (B) Image of P. citreus and E. coli expressing p450cam aer 48h of co-cultivation (described in
methods). (B) was taken using the Texas Red lter on a Zeiss AxioImager M1 (excitation/emission 561/615).

Vol.:(0123456789)
SCIENTIFIC REPORTS
| (2020) 10:15091 |
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visible in the media. Aer D
5
, the population of E. coli DH5α had declined. Instead, a number of diverse native
soil bacteria now contained the plasmid, a trend which continued over the course of the experiment (Fig.5).
Based on morphological analysis of over 100 microscopy images and pairwise mating between bacteria isolated
and identied (via sequencing) from Shell Pond, these bacteria belonged to the Pseudomonas, Flavobacteria,
and Actinomycete genera among others. Plating out aliquots of soil at regular time points conrmed the data
gathered by microscopy: the number and diversity of bacteria expressing the plasmid increased 50-fold over
the rst 30days of the experiment (from 2.6 × 10
–4
CFU at D
0
to 1.25 × 10
–6
CFU at D
30
) (SI Fig.11). e spider-
silk-like biolms formed by native soil microbiota present in the soil were also uorescent (Fig.5), suggesting
the p450cam enzymes also play a role in extracellular degradation of petroleum hydrocarbons under real-world
conditions. GC/MS analysis showed that the amount of total petroleum hydrocarbons within the contaminated
sediments decreased by 46% within 60days compared to untreated soil samples. We le the experiment running
and aer 120days bacteria carrying the vector were still prolic (SI Fig.12). A pilot experiment using articially
contaminated water samples suggest that genes are transferred from E. coli to indigenous bacteria only when
oil is present. In the control samples without oil, we saw no bacteria carrying the vector over the course of the
30-day experiment (the experiment will be published fully in a later publication).
HGT is thought to play a role in the degradation of environmental toxins
53
and several studies have shown
that wild-type bacteria carrying large plasmids with degradative genes can pass these genes on to a limited num-
ber of bacteria
54
. However, this is the rst study to provide evidence for HGT between E. coli DH5α carrying a
small, non-conjugative vector and wild soil microbiota. Our results show that adding engineered E. coli DH5α
carrying small synthetic plasmids to polluted environmental samples may be even more eective than adding
a wild-type bacteria with a larger catabolic vector. Previous studies show that frequencies of HGT between the
donor and recipient bacteria in soil are low (e.g. 3 × 10
–3
CFU transconjugants per gram of sterile soil), recipient
cells come from only a few genera, and the spread of the catabolic vector through the microbial community
does not always lead to enhanced degradation
5557
. e high transfer frequency of synthetic plasmids like the
ones used in this study could be due to several reasons, including the small size of the vector (natural plasmids
Figure4. TEM conrmation of horizontal gene transfer among E. coli expressing pSF-OXB15-p450camfusion
and P. putida. (A) E. coli DH5α (E) connected to P. putida (P) via conjugative pili. (B) Mating-pair bridge
between P. putida and E. coli DH5α. (C) E. coli DH5α with conjugative pili. (D) Nanotube connecting P. putida
and E. coli DH5α.

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References
More filters
Book ChapterDOI
01 Jan 2011
TL;DR: In-situ burning of oil spills is recognized as a viable alternative for cleaning up oil spills on land and water as mentioned in this paper, which can rapidly reduce the volume of spilled oil and eliminate the need to collect, store, transport, and dispose of recovered oil.
Abstract: In-situ burning is recognized as a viable alternative for cleaning up oil spills on land and water. When performed under the right conditions, in-situ burning can rapidly reduce the volume of spilled oil and eliminate the need to collect, store, transport, and dispose of recovered oil. In-situ burning can shorten the response time to an oil spill, thus reducing the chances that the oil will spread on the water surface and thereby aiding in environmental protection. Such rapid removal of oil can also prevent the oil from reaching shorelines, which are difficult to clean. What remains after an in-situ burn are burn by-products such as carbon dioxide, water, some smoke particulate, and unburned oil (residue). Sufficient information is now available to predict levels of these emissions and calculate safe distances downwind of the fire. This chapter contains a compilation of information about in-situ burning of oil spills and includes the scientific aspects of the burning process and its effects, examples from the extensive research into in-situ burns, and practical information about the procedures to be followed and equipment required for carrying out an in-situ burn.

37 citations

BookDOI
01 Jan 2012
TL;DR: This book discusses the development and deregulation of the Plum Pox Virus resistant transgenic plum 'HoneySweet', and the Globalization of Agricultural Biotechnology: Implications for Regulatory Compliance, Stewardship and Stakeholder Engagement.
Abstract: Introduction.- Foreword.- 1. Introduction to Biotechnology Regulation: A U.S. History.- 2. Regulation of genetically modified crops in USA and Canada: Canadian overview.- 3. Regulation of genetically modified crops in USA and Canada: American overview.- 4. Regulation of Genetically Engineered Microorganisms under PPA, FIFRA and TSCA.- 5. The promise and potential perils of genetically modified microorganisms in agriculture and the environment.- 6. Regulatory experiences in symbiotic control of Pierce's disease.- 7. Over a Decade of Crop Transgenes Out-of-place.- 8. The Regulation of Organisms used in Agriculture under the Canadian Environmental Protection Act, 1999.- 9. Regulating the Environmental Release of Plants with Novel Traits in Canada.- 10. Regulatory Requirements for Plant-Incorporated Protectants.- 11. United States Environmental Protection Agency Insect Resistance Management Programs for Plant-Incorporated Protectants and Use of Simulation Modeling.- 12. Development and deregulation of the Plum Pox Virus resistant transgenic plum 'HoneySweet'.13. Genetically Engineered Insects - Regulatory Progress and Challenges.- 14. Regulation of Genetically Engineered Animals.- 15. Regulatory science, research science and safety assessment in agricultural biotechnology.- 16. The Globalization of Agricultural Biotechnology: Implications for Regulatory Compliance, Stewardship and Stakeholder Engagement.- 17 - Facilitating market access for GE crops developed through public sector research.

34 citations

Book ChapterDOI
01 Jan 2012
TL;DR: The focus of this chapter is the regulatory process for approval of the use of genetically engineered microbes under the oversight of the U.S. EPA and instances where organisms may be exempted from oversight and the outlook for the application of GE microbes in the future.
Abstract: Since the dawn of civilization, humans have utilized microbial organisms of various sorts for food and agricultural production. More recently, microbes have been used for pesticidal, and environmental management purposes. With the advent of the development of recombinant DNA technology to genetically alter microbes, it became necessary for Federal regulators to assess the appropriate level, format, and application of their regulatory authorities. In 1986, the Office of Science and Technology Policy issued the Coordinated Framework for Regulation of Biotechnology. The Coordinated Framework constituted a comprehensive regulatory policy for biotechnology that, in essence, concluded that no new statutory authorities were necessary to effectuate a robust and efficient regulatory program for the products of biotechnology. The Framework articulated a division of regulatory responsibilities for the various agencies then involved with agricultural, food, and pesticidal products. Thus, in accordance with the Framework, USDA APHIS regulates microbes that are plant pests under the Plant Protection Act (PPA) and the National Environmental Policy Act (NEPA); the U.S. Environmental Protection Agency (U.S. EPA) regulates microorganisms and other genetically engineered constructs intended for pesticidal purposes and subject to the Federal Insecticide Fungicide and Rodenticide Act (FIFRA) and the Federal Food Drug and Cosmetic Act (FFDCA). The U.S. EPA also regulates certain genetically engineered microorganisms used as biofertilizers, bioremediation agents, and for the production of various industrial compounds including biofuels under the Toxic Substances Control Act (TSCA). The focus of this chapter is the regulatory process for approval of the use of genetically engineered microbes under the oversight of the U.S. EPA. We will also consider instances where organisms may be exempted from oversight and the outlook for the application of GE microbes in the future. This chapter does not seek to serve as a guidebook for navigating the details of the regulatory process, but rather as an overview of key considerations in risk assessment and risk management.

32 citations

Frequently Asked Questions (16)
Q1. What are the contributions in "Horizontal ‘gene drives’ harness indigenous bacteria for bioremediation" ?

In this paper, the authors developed a new technology to harness indigenous soil microbial communities for bioremediation by flooding local populations with catabolic genes for petroleum hydrocarbon degradation. 

Publicly available documentation and potentially even de-centralized approval of GM field trials ( e. g. through university Environmental Health and Safety offices ) could make field trials of GM bacteria more achievable in the near future. Future research is needed to determine ( 1 ) how long these plasmids are maintained under field conditions, ( 2 ) whether genetic mutations accumulate over time that might impact enzyme functioning, and ( 3 ) how vector-based gene drives harnessing natural processes of conjugation may affect local microbial community composition and soil metabolic functions. 

Replacing antibiotic selection markers with chromoprotein ones64 would eliminate the release of antibiotic resistance genes into the environment. 

E. coli DH5α engineered to carry plasmids containing genes involved in degradation of environmental toxins could be used to augment the capacity of native soil microbial communities to degrade pollutants of interest. 

Current approaches to removing crude oil from the environment include chemical oxidation, soil removal, soil capping, incineration, and oil skimming (in marine contexts)7,8. 

Practices such as skimming only remove the surface fraction of the oil while the water-soluble portion cannot be recovered, negatively affecting marine ecosystems12,13. 

Previous studies show that frequencies of HGT between the donor and recipient bacteria in soil are low (e.g. 3 × 10–3 CFU transconjugants per gram of sterile soil), recipient cells come from only a few genera, and the spread of the catabolic vector through the microbial community does not always lead to enhanced degradation55–57. 

The primary barriers to implementing this approach on current polluted industrial sites are (1) lack of standardized procedures to test and ultimately allow the use of GM organisms for environmental applications and (2) the willingness of site managers to adopt this approach to remediation. 

To determine the survival rate of engineered bacteria in contaminated soils, the authors added E. coli containing the plasmid pSF-OXB15-p450camfusion to sediment taken from a former Shell refinery in Bay Point, CA. 

Dubey and Ben-Yehuda52 show in their classic paper that gfp molecules, calcein, and plasmids could be transferred between B. subtilis cells. 

To determine whether their engineered bacteria could transfer non-conjugative, synthetic vectors containing petroleum-degrading genes to indigenous soil and marine bacteria, the authors conducted a series of mating experiments. 

The second barrier can be overcome through public engagement with those working in the remediation sector (industry, site managers, and remediation consulting firms) and a shift in their approach to how the authors conduct remediation (favoring slower biological-based solutions that harness local ecological and chemical processes over faster processes such as oxidation and soil removal). 

They also show that a non-integrative vector carrying a resistance marker from B. subtilis could be transferred to Staphylococcus aureus and E. coli (with recipient cells expressing antibiotic resistance). 

Solid-Phase MicroExtraction (SPME) GC/MS analysis of these cultures revealed that all three bacteria degraded 99% of dodecane in 10 days. 

To determine whether HGT of the synthetic vectors to wild-type bacteria was achieved through mating, the authors conducted TEM of wild-type bacteria after exposure to E. coli DH5α carrying pSF-OXB15-p450camfusion. 

Fluorescence microscopy also revealed for the first time the key role of extracellular enzymes in degradation of petroleum hydrocarbons.