Plasmid 60 (2008) 38–44

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Plasmid
journal homepage: www.elsevier.com/locate/yplas

Review

Bacterial conjugation-based antimicrobial agents

Marcin Filutowicz a,*, Richard Burgess b, Richard L. Gamelli d, Jack A. Heinemann c,
Brigitta Kurenbach c, Sheryl A. Rakowski a, Ravi Shankar d
a
Department of Bacteriology, University of Wisconsin, Microbial Sciences Building, 1550 Linden Dr., Madison, WI 53706, USA
b
McArdle Laboratory For Cancer Research, University of Wisconsin, Madison, WI, USA
c
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
d
Department of Surgery, Loyola University Medical Center, Maywood, IL, USA

a r t i c l e i n f o a b s t r a c t

Article history:
Received 18 March 2008
Available online 14 May 2008
We dedicate this review to the memory of
Joshua Lederberg for his insights into the
mating habits of bacteria and many other
ground-breaking discoveries. ‘‘I propose
plasmid as a generic term for any
extra-chromosomal hereditary determinant.’’
J. Lederberg (1952).
A clear imperative exists to generate radically different antibacterial technologies that will
reduce the usage of conventional chemical antibiotics. Here we trace one route into this
new frontier of drug discovery, a concept that we call the bacterial conjugation-based tech-
nologies (BCBT). One of the objectives of the BCBT is to exploit plasmid biology for combat-
ing the rising tide of antibiotic-resistant bacteria. Specifically, the concept utilizes
conjugationally delivered plasmids as antimicrobial agents, and it builds on the accumu-
lated work of many scientists dating back to the discoveries of conjugation and plasmids
themselves. Each of the individual components that comprise the approach has been dem-
onstrated to be feasible. We discuss the properties of bacterial plasmids to be employed in
BCBT.
Ó 2008 Elsevier Inc. All rights reserved.

Communicated by Dhruba K. Chattoraj

Keywords:
Antimicrobials
Bacterial conjugation
Biotherapeutics
Antibiotic resistance

1. Introduction 1.1. Threats from antibiotic-resistant bacteria

From its inception as a field of scientific study, bacterial
genetics found plasmids to be instrumental in establishing
the founding principles of molecular biology (reviewed by
Cohen, 1993; Helinski, 2004). With Escherichia coli as the
factory, plasmids became both the machinery and the
products of the recombinant DNA revolution. Today, plas-
mids remain among our most useful tools for sending bio-
logical information from test tubes back to genomes. They
have the potential to be the conduits we use to communi-
cate with the massive populations of microorganisms that
dominate life forms.
* Corresponding author. Fax: +1 608 262 9865.
E-mail address: msfiluto@wisc.edu (M. Filutowicz).
Over the last 50 years, antibiotics have been the main
treatment modality used to fight bacterial infections. Pro-
longed and indiscriminate use of antibiotics to treat infec-
tions in both man and animals has resulted in the rapid
emergence of antibiotic-resistant bacteria. No single drug
is effective against all pathogenic bacteria, and far more
disturbing is the expanding list of bacterial pathogens that
have acquired multiple mechanisms of resistance. Com-
pounding this dire situation, the discovery of new classes
of antimicrobial agents with broad-spectrum antimicrobial
properties has been slow (Talbot et al., 2006). After three
decades of hiatus, only three new classes of antimicrobials
represented by daptomycin, linezolid and tigecycline have
been approved by the FDA since 2000. Significantly, resis-

0147-619X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.plasmid.2008.03.004
 M. Filutowicz et al. / Plasmid 60 (2008) 38–44
tance to these drugs has already been identified in strains
of Acinetobacter baumannii, Pseudomonas aeruginosa,
Enterococcus faecalis and Staphylocoocus aureus (Bour-
geois-Nicolaos et al., 2007; Dean et al., 2003; Montero
et al., 2008; Navon-Venezia et al., 2007).
Analyses of epidemiological data have led to an esti-
mate that the rate of sepsis in the United States will in-
crease by approximately 2% above population growth in
the next 30 years (Angus et al., 2001; Angus and Wax,
2001). This finding underscores the sense of urgency that
exists, not only with respect to developing additional clas-
ses of antibacterial compounds but also for generating
therapeutic approaches based on innovative technologies
and thought processes. Fortunately, our growing appreci-
ation of microbial evolution brings with it the potential
for new kinds of antimicrobial discovery strategies that
might lead to new kinds of treatments. If we allow our
understanding of resistance-acquisition to transform our
pursuit of next-generation antimicrobial agents, the end
products could retain their efficacy longer than conven-
tional antibiotics. In fact, such agents might be the only
way for medicine to keep pace with the rapid acquisition
of antibiotic-resistance by many pathogenic bacteria.
Unfortunately, truly novel antimicrobials could also prove
to be more expensive to produce and more difficult to ac-
cept; and they may require faster diagnoses in order to be
effective.
Screening for compounds that inhibit bacterial growth
when cells are exposed to them has been the standard
operating procedure employed to discover most classes
of antimicrobial agents. This approach has been a produc-
tive one, but it has also limited the breadth of compounds
discovered and employed. Only a small number of drug
classes have been described that are able to penetrate bac-
terial cells and then remain inside them long enough to in-
hibit an essential target molecule. The use of BCBT for
antibiotic delivery circumvents this permeability barrier.
It allows inhibitory compounds (e.g. bacteriocidal proteins
and RNAs) to be expressed directly from plasmids
transferred into unwanted target bacteria.
1.2. Bacterial conjugation
Bacterial conjugation was first observed, in E. coli, by
Joshua Lederberg (Lederberg and Tatum, 1946). We will
limit our discussion of conjugation systems to those of
Gram-negative bacteria, which differ from the analogous
systems of Streptomyces and Gram-positive bacteria (Cle-
well and Francia, 2004; Lawley et al., 2004). Conjugation
systems have three essential components: the transfero-
some (type IV secretion), which spans the cell envelope
and is responsible for the synthesis of the conjugative pilus;
the relaxosome, which is a complex of proteins that process
the DNA at the origin of transfer (oriT); and the coupling
protein, which connects the two other entities together.
During conjugation, a unique plasmid DNA strand called
the transfer (T) strand undergoes 50 -to-30 directional trans-
fer from a donor bacterium to a recipient cell. This T-strand
is cleaved in the donor by a specific nuclease at the nic site
within the oriT. Cleavage is mediated by a plasmid-encoded
relaxase enzyme, which establishes a covalent linkage with
39
the 50 terminus of the T-strand, thereby producing a
covalent protein–DNA transfer intermediate (reviewed by
Lanka and Wilkins, 1995; Lawley et al., 2004; Zechner
et al., 2000). For productive conjugation, a mating pair for-
mation system (Mpf) facilitates cell-to-cell contact. Once
contact is established, T-strand DNA is transferred from do-
nor to recipient in a process that utilizes a type IV secretion
system (Christie et al., 2005; Christie and Vogel, 2000;
Ferguson and Heinemann, 2002; Lawley et al., 2004).
2. Antimicrobials delivered by bacterial conjugation
The conjugative transfer and transposition of DNA are
key phenomena that facilitate the spread of antibiotic
resistance genes, indiscriminately, to beneficial and patho-
genic bacteria alike. This disconcerting observation, how-
ever, eventually led to a provocative question—would it
be possible to turn conjugation, selectively, against the patho-
gens? We would argue that it is indeed possible. The way to
achieve success is by developing antimicrobials that can be
delivered directly from one bacterium to another bacte-
rium utilizing conjugation, a process that can be remark-
ably efficient (Andrup and Andersen, 1999; Andrup et al.,
1998; Jensen et al., 1996; Lawley et al., 2004). The potential
for bacterial plasmids to be used as agents controlling dis-
ease is based on a universal property of conjugative sys-
tems: Plasmid-encoded information, even information
encoding self-destruction, is expressed upon conjugal
transfer of plasmid DNA to a recipient cell (Diaz et al.,
1994; Peng et al., 2006). Surprisingly, the concept of using
conjugation-delivered plasmids as antibacterial agents has
long been overlooked despite the development of a conju-
gation-based technology to limit the lateral spread of
cloned genes (Diaz et al., 1994). Nonetheless, the fact that
bacteria can be killed by a toxin delivered through inter-
and intra-species conjugation has been documented (Hau-
gan et al., 1995; Rodriguez et al., 1995).
2.1. Killing bacteria by unabated replication
Generally speaking, plasmids tend to be circular DNA
molecules that are maintained at a fixed copy number in-
side the bacterial cell. The plasmid’s sound survival strat-
egy is to adjust its own rate of reproduction in
accordance with the reproduction of its host. If the plasmid
reproduces too slowly it will be lost from the bacterial pop-
ulation. If it reproduces too fast, the growth of host bacte-
ria can be arrested by the copious amounts of plasmid
DNA. As a result, plasmid copy number is typically con-
trolled by repressor-like molecules encoded by the plasmid
DNA itself (reviewed in Refs. Chattoraj, 2000; Filutowicz
et al., 1994; Filutowicz and Rakowski, 1998; Krüger et al.,
2004). And where there is a DNA-encoded regulator, there
is an open door allowing researchers to purposefully
manipulate that regulation through mutation. Indeed,
known mutations that disrupt repressor function cause
over-replication of plasmids, producing what is known as
a copy-up phenotype (Stalker et al., 1983). In extreme
cases, copy-up mutations result in runaway plasmid repli-
cation due to the loss of copy-control mechanisms (Blasina
40 M. Filutowicz et al. / Plasmid 60 (2008) 38–44

et al., 1996; Haugan et al., 1995; Molin et al., 1980; Toukd-
arian and Helinski, 1998). In the next step towards the
development of conjugally-delivered antimicrobial agents,
Peng et al. (2006) constructed mobilizable ‘runaway plas-
mids’ that can be maintained in specially engineered donor
cells (see Fig. 1, top panel). Using this system, they demon-
strated that the hyper-replicating plasmids express antimi-
crobial activity upon conjugal transfer to recipient cells
from several bacterial species.
Remarkably, replicative killing of bacteria by copy-up
plasmids appears to be species-specific. For example, the
same copy-up variant that confers only a modest increase
in the copy number of an RK2 derivative plasmid in E. coli
resulted in the replicative killing of P. aeruginosa (Haugan
et al., 1995). Likewise, a copy-up derivative of R6K, plasmid
pFL129, appears to generate a lethal copy number in two
Erwinia species (herbicola and amylovora) but not in
E. coli (Wild et al., 2004).
The concept of employing over-replicating plasmids as
antibacterial ‘‘agents’’ grew from our curiosity about the
possible mechanism(s) by which such plasmids inhibit cell
growth. Our front-runner hypothesis is that runaway rep-
lication of a plasmid acts like a trap to capture all of the
cell’s available replication machinery to the exclusion of
chromosomal replication (Peng et al., 2006). This hypothe-
sis is grounded in a tenet that the synthesis of plasmids
and the synthesis of chromosomes overlap extensively in
their use of cellular DNA replication machinery. As a result,
the acquisition of resistance to runaway plasmid replica-
tion might carry with it a strong selective disadvantage be-

Donor Recipient
mob oriV
ori e
tra
ssion om oriV
repre selective os oriV
marker m
oriV
DNA transfer ro
Ch oriV
oriV
oriT
by conjugation oriV
oriV oriV
Transmissible oriV
Chromosome Plasmid

Runaway Replication Repressed Runaway Replication Occurs

VIABLE KILLED
Donor Recipient
mob
tra e
om
selective
os
marker
anti-kill oriV
DNA transfer m oriV
ro
oriT Ch
kill by conjugation
kill
Transmissible
Chromosome Plasmid

Lethal Function Neutralized Lethal Function Active

VIABLE KILLED
Donor Recipient
mob
tra oriT e
PR kill om kill
repressor
DNA transfer os
oriV
r om
PR Ch oriV
selective by conjugation
marker
PR
Transmissible
Chromosome Plasmid

Lethal Function Repressed Lethal Function Expressed

VIABLE KILLED
Fig. 1. Applications of BCBT. Top: An example of killing bacteria by an engineered over-replicating plasmid (from Peng et al., 2006). Repression of ori
function is achieved by integrating a wildtype rep gene into the donor’s chromosome (red/blue rectangle). Middle: An example of using the interaction
between ‘‘kill’’ (white half circles) and ‘‘anti-kill’’ (black half-circles) to protect the donor from self-distruction. The anti-kill gene (black rectangle) is
integrated into chromosome while the kill gene (white rectangle) is present on the plasmid. Bottom: An example of using repressor/operator interactions to
control expression of a toxic product in a donor. Repression of the PR promoter is achieved by integrating a PR repressor (red half-circles) gene into the
donor’s chromosome (red rectangle). A toxin gene present on the plasmid is indicated by a white rectangle. Conjugational elements, tra and mob, are
indicated. Replication and transfer origins are noted as oriV and oriT, respectively.
 M. Filutowicz et al. / Plasmid 60 (2008) 38–44
cause host mutations that would halt plasmid replication
will compromise replication of the chromosome as well.
Thus resistance to runaway replication based killing might
present far less of a problem than we have encountered
with the occurrence and horizontal spread of resistance
to conventional antibiotics.
2.2. Natural and synthetic products with the potential to be
used as conjugally-delivered antimicrobial agents
Over-replication of plasmid DNA is only one of many
potential antimicrobial agents amenable to BCBT. The pro-
duction of plasmid- or chromosome-encoded bacteriocins
(microcins) is well established (Oppegard et al., 2007;
Severinov et al., 2007). In almost all instances, cells pro-
ducing a bacteriocin also produce a bacteriocin-specific
antidote, typically a peptide or RNA (reviewed in Engel-
berg-Kulka and Glaser, 1999; Gerdes et al., 1986; Kawano
et al., 2007). For BCBT, an anti-kill antidote, which can
neutralize the expression of a plasmid-encoded antimicro-
bial agent, can be integrated into the chromosome of the
donor bacteria (see Fig. 1, middle panel); susceptible
recipients are killed after plasmid transfer from the pro-
tected donor cells. In another approach, a donor might
be rendered insensitive to a killer plasmid by using a
tightly regulatable promoter-operator system in which
the expression of a lethal bacteriocin gene is prevented
by a repressor made only in the donor cell (Anthony
et al., 2004; Bowers et al., 2004 and bottom panel of
Fig. 1). An engineered example of a plasmid with multiple
toxins that are independently regulated has been built
and employed in the proof-of-concept experiments that
are described next.
2.3. BCBT in treating wound infections
ConjuGon Inc. (Madison, WI), and the Loyola University
Medical Center’s Burn and Shock Trauma Institute (May-
wood, IL), have recently reported success in eradicating A.
baumannii in vitro and in an in vivo murine burn sepsis
model (Shankar et al., 2007). A. baumannii is a Gram-nega-
tive opportunistic human pathogen that is found in soil
and water (Hujer et al., 2006) and is easily transmitted in
health care settings. Infections attributable to this patho-
gen are on the rise and present an unmanageable challenge
to medical personnel, particularly in burn and shock trau-
ma units. Wounds such as burns are routinely treated with
topical antibiotics at high enough doses to achieve thera-
peutic concentration, however, such antibiotic treatment
is compromised if the wound is infected with multi-
drug-resistant bacterial strains. The threat of developing
lethal sepsis is intensified. Many clinically-isolated strains
of A. baumannii are pan resistant (resistant to all antibiot-
ics) and the incidence of nosocomial infections caused by
such strains is increasing in critically injured and immuno-
compromised patients who are hospitalized for prolonged
periods (Levin et al., 1999; Perez et al., 2007; Van Looveren
and Goossens, 2004).
Clearly, there is an urgent need for novel therapeutics
that will target A. baumannii and other antibiotic-resistant
pathogens. Work by Shankar et al. (2007) demonstrates the
41
great promise of BCBT in this arena. Genes encoding highly
potent antibacterial proteins were cloned into a plasmid
and maintained in an attenuated E. coli host that ‘‘turned
off’’ expression of the antibacterial agent (outlined in the
bottom panel of Fig. 1). In contrast, the pan-resistant strain
of A. baumannii that served as the target organism in these
experiments lacked the ability to keep these genes ‘‘turned
off.’’ The delivery of the mobilizable plasmid and subse-
quent expression of bacteriocidal proteins in the pathogen
improved the survival of septic mice and reduced A. bau-
mannii colony counts at the burn wound sites.
3. Manipulating conjugation efficiencies to optimize
BCBT
Conjugation efficiency is the measurement of the pro-
ductivity of a conjugation system and it is typically ex-
pressed in terms of the transfer frequency of a plasmid.
For conjugation to be developed into a meaningful delivery
system for antimicrobial agents, high conjugation efficien-
cies would be necessary. It is significant then that many
conjugation systems have two important characteristics
in common. They are able to sustain conjugative DNA
transfer in liquid and solid media and, often, close to
100% of recipient cells acquire the plasmid (Andrup and
Andersen, 1999; Andrup et al., 1998; Jensen et al., 1996).
3.1. The role of restriction-modification systems in
conjugation efficiency
Restriction modification activities are ubiquitous in bac-
teria and may influence the efficacy of BCBT. Some such
systems are plasmid-encoded, while others reside on the
bacterial chromosome (Roberts and Macelis, 1996).
Regardless of their source, these systems employ restric-
tion enzymes to cleave double-strand DNA when a cognate
enzyme has not modified it. In this way, cells can be pro-
tected from an invasion by foreign DNA. Although the
DNA that is transferred during conjugation is single-strand
(resistant to cleavage), there is a race to protect or restrict
as the T-strand is converted to double strand plasmid DNA.
Specific strategies that tackle this potential obstacle will
likely need to be employed for the BCBT to become a prac-
tical antimicrobial technology.
Conveniently, known aspects of plasmid biology in sev-
eral well-studied systems should come in handy in
addressing this important issue (discussed in Wilkins,
2002). For instance, many conjugative plasmids contain
anti-restriction loci (ard) as part of the so-called ‘‘leading
region’’ defined as the first portion of the plasmid to enter
the recipient (Zechner et al., 2000). The products of these
genes act specifically to alleviate restriction of DNA by type
I restriction enzymes. Having an ardA locus present, in cis,
allows an incoming unmodified plasmid to evade restric-
tion when transferred by conjugation (but not by transfor-
mation or electroporation). Protection requires expression
of the incoming gene in the recipient cell (Read et al.,
1992). Moreover, ardA expression is enhanced by the
gene’s conjugative transport into the recipient cell
(Althorpe et al., 1999). Future applications of BCBT will al-
42 M. Filutowicz et al. / Plasmid 60 (2008) 38–44
most certainly capitalize on observations like these by
incorporating one or more anti-restriction systems into
‘killer plasmids’. As an additional benefit, this will provide
researchers yet another tool that can be used to modulate
the antibacterial specificity and efficacy of plasmid/donor
pairs.
3.2. Conjugation in the intracellular milieu of pathogen-
invaded cells
BCBT could perhaps be especially suitable for use inside
eukaryotic cells that have been infected by invasive patho-
gens. These bacteria establish themselves in the intracellu-
lar milieu of their host, thereby evading administered
antibiotics as well as the host’s immune system (Finlay
and Falkow, 1997; Rakita, 1998). Eukaryotic cells have pro-
ven to be a suitable environment for intra-species conjuga-
tive plasmid transfer (in Salmonella enterica) and also for
inter-species conjugation involving Salmonella and other
bacterial species (Ferguson and Heinemann, 2002 and
Kurenbach and Heinemann, manuscript in preparation).
The latter is of particular relevance because cells that are
invaded by Salmonella may subsequently be able to take
up other, naturally non-invasive species (Francis et al.,
1993; Okada et al., 1998).
Plasmids are not the only type of mobile genetic ele-
ment of course. Bacteriophage also fall into this category,
making research exploring their use against intracellular
pathogens relevant to a discussion of BCBT. When benign
Mycobacterium smegmatis was utilized to deliver myco-
phage to intracellular Mycobacterium tuberculosis and
Mycobacterium avium, a reduction of the pathogens by
80% was observed (Broxmeyer et al., 2002). A similar
experiment was conducted in vivo by Danelishvili et al.
(2006) in a mouse model of infection. In their study, the
data demonstrated that a single dose of mycophage corre-
lated with a 50% reduction of pathogenic M. avium, but
only when delivered by M. smegmatis; phage alone had
no such effect. We note that the properties of phage and
plasmids can be easily interchanged as exemplified by
phage P1, which exists as a plasmid in its prophage form
(Ikeda and Tomizawa, 1968).
Table 1
Human health and agricultural applications of live bacteria
Category of use Species
Applications of live bacteria: human health and wellness
Vaccines— Salmonella typhi, Mycobacterium bovis
cellular
Cancer/tumor Mycobacterium bovis
therapy
Applications of live bacteria: animal health and productivity
Vaccines— More than a dozen genera of bacteria are USDA
Cellular licensed
Competitive PreemptÒ—Blend of 29 species
inhibitors
Applications of live bacteria: plant health and productivity
Antifungals Bacillus (3 species), Pseudomonas (3 spp.), Streptomyces
(3 spp.), Serratia plymuthica
Insecticides Bacillus (3 species)
The information in Table 1 is taken from the following web sites: fda.gov, clinicaltrials.gov, aphis.usda.gov, ars.usda.gov, and epa.gov.
Even if highly optimized, it is unlikely that the applica-
tion of BCBT will eliminate 100% of the harmful, targeted
bacteria in an environment. Importantly, however, it is
universally acknowledged that no clinically used antibiotic
can achieve complete pathogen eradication in vivo, yet
conventional antibiotics still cure patients. Clearly, 100%
lethality is not necessary for an antimicrobial method to
be exceedingly valuable. With conventional antibiotics,
the subpopulation of bacteria that survives therapy is typ-
ically eliminated by the host’s immune system. We expect
BCBT to behave similarly to conventional antibiotics in this
regard. Significant reductions in the number of bacteria
colonizing a surface will be sufficient to keep titers of bac-
teria below pathogenic levels in most medical, veterinary
and agricultural applications of the BCBT. Moreover, there
are qualities of therapeutics other than lethality that con-
tribute to efficacy (Heinemann and Goven, 2007). Drugs
with longer useful lives or fewer side-effects might prove
as beneficial in certain applications as drugs that kill more
effectively (Heinemann et al., 2000).
4. Bacteria as therapeutics
Although much work remains to be done to transition
the BCBT from the laboratory bench to real world applica-
tions, the largest obstacle to successful implementation is
unlikely to be a matter of scientific design. The ultimate
challenge may be to demonstrate the safety of the technol-
ogy, both as a medicine and as a new contribution to the
genetically modified organisms in our environment. None-
theless, the proposed use of live bacteria as therapeutic
agents is not as radical as it may seem. Table 1 summarizes
an assortment of applications of live bacteria approved by
U.S. government agencies for use or further study.
Regardless of the pace with which the public accepts
bioengineering in return for its demonstrated benefits,
the potential risks associated with any technology that
uses DNA and/or genetically modified organisms should
not be overlooked. As the BCBT are being developed, the
means to minimize those risks also needs to be explored.
One idea that merits consideration is to take ‘living cells’
out of the equation when using bacteria as the transport
Category of use Species
Probiotics/ Bacillus clausii, Lactobacillus fermentum, Lactobacillus
Prebiotics rhamnosus, Escherichia coli
Probiotics USDA- Bifidobacterium lactis, Lactobacillus rhamnosus
research Streptococcus and Lactococcus
Antinematodal Pseudomonas synxantha
Antibacterials Agrobacterium radiobacter, Pantoea agglomerans
Growth promotion Bacillus (2 species), Pseudomonas fluorescens,
Serratia plymuthic
 M. Filutowicz et al. / Plasmid 60 (2008) 38–44
vehicle for conjugally-delivered antimicrobial agents. Spe-
cifically, BCBT could make use of metabolically active but
non-viable bacterial cells such as minicells and/or maxi-
cells, which are derivatives of mutant bacteria that have
lost their chromosomes due to asymmetric cell division
or degradation, respectively (Frazer and Curtiss, 1975;
Sancar et al., 1979). Despite the absence of the chromo-
some, minicells and maxicells that carry conjugative plas-
mids are capable of conjugal replication and they transfer
plasmid DNA to living recipient cells (Debbia, 1992;
Heinemann and Ankenbauer, 1993; Levy and Norman,
1970). Furthermore cells killed by streptomycin, which
are not compromised in their DNA repair systems, remain
conjugally active (Cavalli-Sforza, 2000; Pardee et al.,
1959). These observations suggest that cells killed by var-
ious mechanisms, except those that cause cells to lyse,
will be conjugation-proficient and, therefore, desirable
tools for certain applications of BCBT.
5. Concluding remarks
The BCBT approach described here is derived from the
efficient transfer of genes that encode various antibacte-
rial agents and relies on the same natural process that
has been ‘gifting’ pathogens with antibiotic resistance
for ages. The use of bacterial conjugation as a drug deliv-
ery system has the potential to avoid many of the pitfalls
of systemic chemical applications (Allen and Cullis,
2004). Once the technology is established, it will need
fine tuning to accommodate different practical applica-
tions. However, with multiple components available for
the tweaking, tailoring the technology for specific targets
should not present insurmountable challenges. Research-
ers can up- or down-regulate plasmid replication, gene
expression and conjugation. We can select donor bacte-
ria based on feasibility and biosafety parameters, or
use conjugation machinery with different host-ranges
to broaden or narrow the activity spectrum as needed.
We can select for mutants or perhaps even engineer do-
nors with unique targeting or survival parameters. It
would seem that the beauty of the BCBT approach is that
there are so many options to explore when it comes to
tailoring the technology to meet specific needs. Thus,
we are convinced that, while unconventional and contro-
versial, antimicrobials delivered by bacteria have the po-
tential to constitute powerful, new alternatives to
conventional antibiotics. Moreover, the pursuit of BCBT
will tremendously enrich our understanding of the con-
jugation process itself, and provide insights into the
forces that influence the trafficking of DNA in the
environment.
Acknowledgments
We thank Hideki Suzuki, Larry Anthony, Donna Bates,
Jennifer Wendt, Miguel Dominguez and Sal Braico, our
present and former colleagues at ConjuGon over the last
few years, for their tireless efforts of translating the ideas
presented in this review into practical applications. We
also thank Don Clewell, James Dahlberg, Jo Handelsman,
43
Denis Maki, Richard Proctor, and Molly Schmid for many
hours of helpful discussions. Additionally, we wish to ex-
press our appreciation to Dr. Joe Dillard for his critical
reading of the manuscript.
Marcin Filutowicz and Richard Burgess are required by
the U.W Conflict of Interest Committee to disclose a finan-
cial interest in ConjuGon. Inc.
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