MINIREVIEW / MINISYNTHE

`
SE
Chemical biology of tetracycline antibiotics
1
Bijan Zakeri and Gerard D. Wright
Abstract: For more than half a century, tetracycline antibiotics have been used to treat infectious disease. However, what
once used to be a commonly prescribed family of antibiotics has now decreased in effectiveness due to wide-spread bacte-
rial resistance. The chemical scaffold of the tetracyclines is a versatile and modifiable structure that is able to interact with
many cellular targets. The recent availability of detailed molecular interactions between tetracycline and its cellular targets,
along with an understanding of the tetracycline biosynthetic pathway, has provided us with a unique opportunity to usher
in a new era of rational drug design. Herein we discuss recent findings that have clarified the mode of action and the bio-
synthetic pathway of tetracyclines and that have shed light on the chemical biology of tetracycline antibiotics.
Key words: antibiotic, tetracycline, biosynthesis, resistance.
Resume : Depuis plus dun demi-sie`cle, les antibiotiques de la famille des tetracyclines ont ete utilises pour traiter les ma-
ladies infectieuses. Cependant, la prescription generalisee de cette famille dantibiotiques en a diminue lefficacite a` cause
dune resistance bacterienne repandue. Lechafaudage chimique des tetracyclines est une structure versatile et modifiable
qui est capable dinteragir avec plusieurs cibles cellulaires. La recente disponibilite de donnees detaillees des interactions
moleculaires de la tetracycline et ses cibles cellulaires conjuguee a` une meilleure comprehension de la voie de biosynthe`se
de la tetracycline nous a fourni une opportunite unique dinaugurer une nouvelle e`re de conception rationnelle de medica-
ments. Nous discutons ci-dessous des recentes decouvertes qui ont permis de clarifier le mode daction et la voie de bio-
synthe`se des tetracyclines, et qui ont fait la lumie`re sur la chimie biologique des antibiotiques de la famille des
tetracyclines.
Mots-cles : antibiotique, tetracycline, biosynthe`se, resistance.
[Traduit par la Redaction]
Introduction
Tetracyclines (Fig. 1) were first discovered in 1948 by
Benjamin Duggar (Duggar 1948) as natural products pro-
duced by species of Streptomyces, and have since proven to
be an economically valuable drug class over the past 6 dec-
ades. The highly modified chemical scaffold of the tetracy-
clines affords them versatility and allows them to interact
with a variety of biological targets. A remarkable feature of
tetracyclines is the presence of keto-enol functional groups
on one face of the scaffold, providing them with the ability
to chelate divalent cations, which plays a prominent role in
their biological functions. Although the chemical biology of
tetracycline antibiotics has been extensively studied, there
are still many aspects of their structureactivity relationships
that are poorly understood.
Streptomyces is a genus of soil-dwelling bacteria that pro-
duce chemically diverse natural products, including tetracy-
clines. As a result, tetracyclines can be easily and cost
effectively isolated by fermentation, which has contributed
to their extensive use in human therapy, veterinary medi-
cine, animal growth promotion, and aquaculture (Chopra
and Roberts 2001). Tetracyclines are broad-spectrum antibi-
otics that act against gram-positive and gram-negative bacte-
ria, the intracellular pathogens chlamydiae, mycoplasmas,
and rickettsiae, as well as eukaryotic protozoan parasites
(Roberts 2003). In addition to their antimicrobial activities,
they have also been prescribed for many noninfectious con-
ditions since the early 1950s. Their clinical applications in-
clude their use as metal chelating ionophores, in the
inhibition of inflammation, in proteolysis, in angiogenesis,
and as anti-apoptotic agents (Ross et al. 1998; Sapadin and
Fleischmajer 2006). Furthermore, this chemical scaffold is
being investigated for its anti-cancer and anti-metastatic po-
tential, in which tetracycline analogues are used as potent
inhibitors of matrix metalloproteinases. The extensive use
of this class of compounds in medicine and agriculture is
paralleled by production levels measured in tons per year.
This has contributed to the emergence of wide-spread bacte-
Received 15 October 2007. Revision received 12 December
2007. Accepted 19 December 2007. Published on the NRC
Research Press Web site at bcb.nrc.ca on 27 March 2008.
B. Zakeri and G.D. Wright.
2
Department of Biochemistry and
Biomedical Sciences, DeGroote School of Medicine, McMaster
University, 1200 Main St. W, Hamilton, ON L8N 3Z5, Canada.
1
This paper is one of a selection of papers published in this
Special Issue, entitled CSBMCB Systems and Chemical
Biology, and has undergone the Journals usual peer review
process.
2
Corresponding author (e-mail: wrightge@mcmaster.ca).
124
Biochem. Cell Biol. 86: 124136 (2008) doi:10.1139/O08-002 # 2008 NRC Canada
rial resistance and a corresponding decline in the use of tet-
racycline antibiotics for human therapy.
Recently many advances have been made in elucidating
the structureactivity relationships, resistance mechanisms,
and biosynthetic pathways of tetracyclines. The availability
of high resolution crystallographic structures of tetracycline
bound to the 30S ribosomal subunit and a detailed under-
standing of the tailoring steps in the biosynthetic pathways
will result in exciting new strategies for the rational design
of novel tetracycline derivatives. Herein we review new
findings that shed light on how tetracyclines interact with
their cellular targets and the secondary metabolic pathways
that lead to their production from simple precursor mole-
cules.
Tetracycline antibiotics
The first members of the tetracycline antibiotics to be de-
scribed, chlortetracycline (CTC) and oxytetracycline (OTC)
(Fig. 1), were discovered in the late 1940s during the early
period of the Golden Age of antibiotic discovery as a result
of natural product screening programs. These compounds
are broad-spectrum antibiotics that act against a plethora of
microbes from diverse physiological, ecological, and genetic
backgrounds, making them attractive to the pharmaceutical
industry. Not only are they relatively easy and inexpensive
to produce, but they do not cause severe side effects and
have favourable oral bioavailability and pharmacokinetic pa-
rameters (Agwuh and MacGowan 2006). As a result of their
impressive bioactivity, tetracyclines were swiftly promoted
through clinical trials (Duggar 1948). All of these attributes
made tetracyclines a favourable choice of drug as a first-line
defence against bacterial infections. Soon thereafter, many
more members of this group of antibiotics were isolated as
fermentation products produced by soil-dwelling organisms,
making tetracyclines a large family of antimicrobials. At
about the same time, Stokstad et al. observed that the inclu-
sion of fermentation products from the CTC producer Strep-
tomyces aureofaciens accelerated the growth of poultry,
which demonstrated the potential of these compounds as
growth promoting agents in the agricultural industry (Stok-
stad et al. 1949). Naturally, the extensive use of tetracy-
Fig. 1. Selected members of the tetracycline family of antibiotics.
Zakeri and Wright 125
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2008 NRC Canada
clines, both in the clinic and in agriculture, imposed a mas-
sive selection pressure for resistant isolates. Not surpris-
ingly, by 1953 the first tetracycline-resistant strains of
Shigella dysenteriae were isolated, and soon afterwards in
1955, multidrug resistant Shigella species that were resistant
to tetracycline, chloramphenicol, and streptomycin were dis-
covered (Watanabe 1963; Roberts 1996).
To keep pace with the rapid emergence of resistant patho-
gens, new tetracyclines were sought out; however, new tet-
racyclines from traditional microbial sources proved
difficult to find. As a result, we entered a golden age of
antibiotic medicinal chemistry, during which, scientists
tried to improve potency and evade resistance mechanisms
by chemically modifying antibiotic scaffolds (Wright 2007).
This resulted in the development of semisynthetic second-
generation tetracyclines, which include doxycycline and
minocycline (Fig. 1). These compounds are more lipophilic
than their parent compounds, and as a result, they have bet-
ter absorption and pharmacokinetic parameters (Agwuh and
MacGowan 2006). Recently, third-generation tetracyclines,
which include the semisynthetic glycylcyclines and amino-
methylcyclines, have been developed (Sum and Petersen
1999; Agwuh and MacGowan 2006), some of which are
currently in clinical trials (e.g., Paratek Pharmaceuticals,
www.paratekpharm.com/pt_tet_inhib.html). Tigecycline is a
member of the glycylcyclines, and was approved for clinical
use by the US Food and Drug Administration in 2005. It
contains an N-alkyl-glycylamido group at position 9 on the
scaffold of minocycline (Fig. 1). This modification results
in a more efficient interaction with the ribosome and the
evasion of classic tetracycline-resistance mechanisms such
as efflux pumps and ribosomal protection proteins (vide in-
fra) (Slover et al. 2007).
Tetracyclines and indeed, many other natural products,
often possess very complex chemical structures that are pro-
duced by a large number of enzymes in a "conveyor-belt"
fashion. Once the scaffolds of these compounds are pro-
duced, many regio-specific tailoring reactions position func-
tional groups at strategic location, allowing the compounds
to interact with biological targets via hydrogen bonding. As
a result, tetracyclines contain several chiral centres and
many chemically reactive substituents, which makes their
total chemical synthesis challenging. Furthermore, their
chemical sensitivity to acidic and basic media, which cause
the molecules to undergo chemical transformations, further
hinders synthetic approaches (Muxfeldt et al. 1979). For
these reasons, it would seem that the ideal method of devel-
oping novel tetracycline derivatives would be through semi-
synthetic approaches. Nevertheless, successful attempts at
the total synthesis of tetracyclines have been made. In 1979,
Muxfeldt et al. (Muxfeldt et al. 1979) reported the total syn-
thesis of OTC from a tetralone aldehyde producing 47 other
precursor molecules before isolating OTC in an unspecified
yield. Further improvements to this approach have been
made recently by Myers group. In 2005 they reported the
synthesis of ()-tetracycline from benzoic acid in 17 steps
with an overall yield of 1.1% (Charest et al. 2005a). In the
same year, they also reported the synthesis of ()-doxycy-
cline, also from benzoic acid, with an 8.3% yield over 18
steps (Charest et al. 2005b). However, total syntheses of
such complex molecules are time consuming, labour inten-
sive, and expensive, which highlights the advantages of
semisynthetic modifications for the improvement of antibiot-
ics.
After *40 years of use in the clinic, tetracyclines began
to decline as first-line antibiotics. This decline correlated
with certain key events. First, in the early 1980s, the mecha-
nism by which the Escherichia coli transposon Tn10 confers
tetracycline resistance was identified. This led to the identi-
fication of several tetracycline-resistance determinants that
functioned by reducing intracellular tetracycline concentra-
tions through active efflux (Ball et al. 1980; McMurry et al.
1980). Second, several members of the fluoroquinolone class
of antimicrobials emerged and received FDA approval, lead-
ing to their subsequent clinical use. Although they function
by inhibiting DNA gyrase activity, the fluoroquinolones,
like the tetracyclines, are broad-spectrum antimicrobials and
were therefore an attractive alternative to tetracyclines.
These events contributed to a general decline over the past
few decades in the clinical use of tetracyclines for the treat-
ment of common infectious diseases.
Nevertheless, tetracyclines are still potent inhibitors of
bacterial protein synthesis and are amenable to chemical
modification. An understanding of the mode of action of tet-
racyclines at the molecular level will no doubt provide use-
ful information that will enable the production of novel
compounds that can bypass resistance mechanisms.
Mode of action
Tetracyclines impart their broad spectrum antibiotic activ-
ities through both bacteriostatic and bactericidal modes of
action. The clinically used compounds are bacteriostatic and
their mode of action has been well characterized, whereas
the action of bactericidal tetracyclines, such as chelocardin
(Fig. 1), is poorly understood (Schnappinger and Hillen
1996). Recently, Kohanski et al. have shown that the cellu-
lar killing capabilities of three major classes of bactericidal
antibiotics (quinolones, b-lactams, and aminoglycosides) can
be partly credited to the production of toxic hydroxyl radi-
cals (Kohanski et al. 2007). Although there is no evidence
of hydroxyl radical formation by bactericidal tetracyclines,
it does warrant further investigation. The antimicrobial func-
tion of tetracycline has been attributed to the binding of the
bacterial 30S ribosomal subunit near the A site and subse-
quent inhibition of protein synthesis by the prevention of
aminoacylated-tRNA docking (Maxwell 1967; Brodersen et
al. 2000). However, there is considerable biochemical evi-
dence for the presence of one strong and several minor bind-
ing sites for tetracycline in the ribosome, which has led to
discrepancies in the mode of action and the functions of the
minor binding sites reported in the literature (Tritton 1977;
Epe and Woolley 1984). The recent revelation of the molec-
ular interactions between tetracycline and the ribosome
through X-ray crystallography has clarified their mode of
action.
Uptake
To gain access to the ribosome, tetracyclines must first
traverse biological membranes and do so in both gram-posi-
tive and gram-negative organisms. It has long been known
that tetracyclines chelate divalent cations, commonly mag-
126 Biochem. Cell Biol. Vol. 86, 2008
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2008 NRC Canada
nesium, and this ionic interaction is pivotal for their cellular
functions (White and Cantor 1971). Likewise, the ionic state
and charge of tetracyclines plays a key role in drug uptake.
The mechanism of tetracycline uptake has been reviewed
elsewhere (Nikaido and Thanassi 1993; Schnappinger and
Hillen 1996) and will therefore not be discussed in detail
here. Briefly, to enter the periplasm of gram-negative bacte-
ria, tetracyclines cross the outer membrane by passing
through porin channels while in complex with Mg
2+
. Subse-
quently, the tetracyclineMg
2+
complex dissociates, thereby
allowing tetracycline to simply diffuse through the cytoplas-
mic membrane to enter the cytosol, where it once again che-
lates an Mg
2+
ion to bind the ribosome (Schnappinger and
Hillen 1996).
Mode of action
Tetracyclineribosomal interactions have been extensively
studied since the 1960s through in vitro biochemical analy-
sis, mechanisms of action of tetracycline derivatives, and
UV-induced cross-linking studies. Yet many unanswered
questions remained as to how tetracycline prevents the bind-
ing of aminoacylated-tRNA to the A site or whether it inter-
acts with the RNA or protein components of the ribosome.
Many of these questions were answered when two groups
independently published detailed crystal structures of tetra-
cycline in complex with the 30S ribosomal subunit of Ther-
mus thermophilus. The first model was published by
Brodersen et al. in 2000 (Brodersen et al. 2000), in which
they found two binding sites for tetracycline on the small ri-
bosomal subunit. Soon thereafter, Pioletti et al. (2001) pub-
lished another structure that depicted six tetracycline binding
sites on the 30S ribosome (named Tet-1 to Tet-6, based on
decreasing relative occupancy) where sites Tet-1 and Tet-5
corresponded to the primary and secondary binding sites of
the Brodersen et al. model, respectively. In both models, the
highest occupied binding site was near the A site, consistent
with the reported mode of action.
The Tet-1 tetracycline binding pocket measures 20 A

wide and 7 A

deep and is located just above the tRNA bind-

ing site between the head and shoulder of the 30S subunit
(Brodersen et al. 2000). Based upon the helix numbering
system proposed by Mueller and Brimacombe (1997), this
site is located in the minor groove of H34 and the stem
loop of H31, where bases 1196 and 1054 point out of H34
and bind tetracycline through hydrophobic interactions
(Pioletti et al. 2001). Once in the binding site, the
tetracyclineMg
2+
complex interacts exclusively with the
16S RNA component of the ribosome and does so through
electrostatic interactions. The hydrophilic side of the mole-
cule, which chelates Mg
2+
, interacts with the backbone
phosphate oxygen atoms of H31 and H34, where the Mg
2+
ion also makes salt bridges (Brodersen et al. 2000). Upon
tetracycline binding, the residues in H31 and H34 undergo
a small conformation change where the gap between the
two head helices slightly increases to accommodate the
molecule. However, there are no other conformation
changes as a result of tetracycline binding, which merely
acts as a physical barrier that blocks the access of tRNA
to the A site (Zarivach et al. 2002). The five minor bind-
ing sites for tetracycline that were observed in the model
of Pioletti et al. all interact with 16S RNA, except for
Tet-2, which binds in a hydrophobic pocket made by a ri-
bosomal protein (Pioletti et al. 2001). Pioletti et al. pro-
posed that these sites may function synergistically with the
Tet-1 to account for the protein inhibitory action of tetra-
cyclines (Pioletti et al. 2001). All of the binding sites, ex-
cept for Tet-3, contact rRNA binding proteins that are
required for the assembly of new 30S subunits, and thus
tetracycline molecule binding may disrupt this process and
inhibit the early steps of protein biosynthesis (Auerbach et
al. 2002; Zarivach et al. 2002). Of particular interest is the
Tet-5 site, where tetracycline makes hydrophilic contacts
with H27 and H11 and is enclosed in a tight pocket sur-
rounded by H20, H27, and S17 (Pioletti et al. 2001). This
site, which was also observed in the Brodersen et al.
model, is interesting because the switch helix H27 is lo-
cated in the decoding centre of the 16S RNA. Along with
H44, H27 undergoes conformational changes to accommo-
date base pairing interactions between tRNA anti-codons
and mRNA codons, which are pivotal interactions in the
decoding process. Tetracycline binding to Tet-5 may limit
these conformation changes, which leads to the disruption
of translation, and hence, the inhibition of protein synthesis
(Auerbach et al. 2002; Zarivach et al. 2002). These de-
tailed accounts of molecular interactions and the location
of the Tet-1 site shed light on the chemical diversity that
is present in the tetracycline group of antibiotics, in which
variations are restricted to the hydrophobic side of the
molecules.
Analysis of the topography of the Tet-1 site in the pres-
ence of tetracycline provides insight into the chemical inter-
actions between tetracyclines and the ribosome (Fig. 2b).
The hydrophilic side of the molecule is responsible for the
majority of the chemical interactions with the 16S RNA
moiety of the ribosome, whereas the more hydrophobic side
appears to be in more open space. This is consistent with
chemical modifications on the hydrophobic side that have
been shown to be well tolerated by tetracyclines while still
maintaining their cell-growth inhibitory properties. Modifi-
cations made to the tetracycline scaffold on C1, C10, C11,
and C12 (Fig. 2a) lead to a loss of antimicrobial activity
(Nelson et al. 1994). On the other hand, modifications can
be made in positions C2, C4, C5, C6, C7, C8, and C9 while
still maintaining the protein synthesis inhibitory activity of
the parent compound, as can be seen among the natural
product compounds of the tetracycline family (Fig. 1)
(Nelson 2002). This molecular model is in agreement with
the bioactive semisynthetic tetracycline derivatives that
have been made. Modifications have been made to tetracy-
cline scaffolds to improve their pharmacokinetic properties,
evade clinically relevant resistance mechanisms, improve
antimicrobial potency, and modify the compounds for me-
dicinal uses other than antibiotics. However, with our cur-
rent understanding, there is potential for a new phase of
tetracycline design in which analogues can be rationally de-
veloped by first modelling them into the ribosomal binding
site, and then modifying them in an attempt to disrupt key
interactions involved in resistance.
Atypical tetracyclines
Certain tetracycline analogues, such as chelocardin and 6-
thiatetracycline, are termed atypical tetracyclines because
Zakeri and Wright 127
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2008 NRC Canada
they are bactericidal drugs (Chopra 1994; Schnappinger and
Hillen 1996). Although these molecules are potent antimi-
crobials with comparable minimal inhibitory concentrations
as their counterparts, they are of little therapeutic value as a
result of their adverse side effects (Rasmussen et al. 1991;
Chopra 1994). When tested against cell-free translation sys-
tems of E. coli and B. subtilis, they were shown to be poor
inhibitors of protein synthesis (Rasmussen et al. 1991). Also,
it was demonstrated that atypical tetracyclines provided little
protection against chemical modification of the 16S RNA
residues that are otherwise protected by tetracyclines, imply-
ing that they do not bind to the conventional tetracycline
binding site in the 30S ribosomal subunit (Rasmussen et al.
1991). Furthermore, when added to growing bacterial cul-
tures, tetracyclines were shown to limit culture growth by
inhibiting the incorporation of amino acids in protein syn-
thesis. However, atypical tetracyclines not only inhibit pro-
tein synthesis but also completely prevent the incorporation
of nucleic acids into DNA and RNA, which led to the theory
that these molecules function by disrupting the cytoplasmic
membrane (Rasmussen et al. 1991). Further evidence for
this notion was provided when it was shown that, in the
presence of atypical tetracyclines, E. coli cytoplasmic mem-
branes undergo morphological alterations that lead to even-
tual cell lysis (Oliva et al. 1992). In solution, tetracyclines
exist as an equilibrium mixture of two species, in which
Fig. 2. (a) A labelled tetracycline compound in complex with Mg
2+
. The highlighted region represents substituents that are required for the
protein inhibitory activity of tetracyclines (Nelson et al. 1994; Nelson 2002). (b) Tetracycline in complex with the 30S ribosomal subunit of
Thermus thermophilus, as determined by Pioletti et al. (PDB file 1I97) (Pioletti et al. 2001). The magnified region in the left panel illus-
trates tetracycline in complex with Mg
2+
(yellow sphere) bound in the Tet-1 binding pocket of the 30S ribosomal subunit. The magnified
region in the right panel illustrates a green tetracycline molecule in complex with a yellow Mg
2+
ion bound to the Tet-1 binding pocket of
the 30S ribosomal subunit of T. thermophilus, and a red nucleotide on the left of the image, which corresponds to the equivalent position of
base 1058 in the 16S rRNA sequence of Escherichia coli. In propionibacteria, a mutation from cytosine to guanine at base 1058 leads to
tetracycline resistance (Ross et al. 1998).
128 Biochem. Cell Biol. Vol. 86, 2008
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2008 NRC Canada
one form is lipophilic and non-ionized while the other is hy-
drophilic and zwitterionic (Hughes et al. 1979). The lipo-
philic form is important during drug uptake, whereas the
hydrophilic form is required for ribosomal binding. The pre-
ferred species of chelocardin, and presumably of the other
atypical tetracyclines, is apparently the lipophilic form,
which along with the planarity of the BCD rings in the mol-
ecules, provides the ideal structure for the compounds to en-
ter the hydrophobic cytoplasmic membrane. They are likely
to be retained once they are in the membrane, where they
cause membrane damage and eventual lysis (Chopra et al.
1992; Chopra 1994; Rasmussen et al. 1991). This may ex-
plain the adverse side effects seen with the therapeutic use
of these compounds, which may be because of indiscrimi-
nate interactions with both prokaryotic and eukaryotic mem-
branes (Chopra et al. 1992). This possible mode of action is
in agreement with the observation that the atypical tetracy-
clines retain their antibiotic activities even in the presence
of the two most common forms tetracycline resistance, drug
efflux and ribosomal protection (Oliva and Chopra 1992).
The protein synthesis inhibitory action of tetracyclines,
combined with a versatile structure that allows them to tra-
verse biological membranes with ease, are key to the broad
spectrum activity of this group of antibiotics. Consequently,
their extensive use has lead to the emergence of wide-spread
resistance among both pathogenic and nonpathogenic bacte-
ria, which has severely crippled the clinical application of
these drugs.
Resistance
Genes that confer tetracycline resistance are among some
of the most commonly found antibiotic resistance determi-
nants in bacteria. They are often located on mobile genetic
elements, such as plasmids and transposons, which can be
passed among bacteria of different genera through horizontal
gene transfer (Speer et al. 1992). Also, common resistance
mechanisms are nondestructive, leading to a prolonged ex-
posure of bacteria to tetracyclines that results in an extended
period of selection for resistant isolates (Hillen and Berens
1994). The nomenclature, mechanisms, and epidemiology of
tetracycline resistance has been extensively reviewed else-
where (Refer to references Connell et al. 2003; Speer et al.
1992; Roberts 2003, 2005; Chopra and Roberts 2001) and
will therefore be only briefly discussed here.
There are four mechanisms by which bacteria become re-
sistant to tetracyclines (Fig. 3): (i) by reducing the intracel-
lular concentration of the compound through active efflux
(Ball et al. 1980), (ii) through disruption of the tetracy-
clineribosomal interaction by ribosomal protection proteins
(RPPs) (Burdett 1991), (iii) by enzymatic inactivation of the
drug through monohydroxylation (Yang et al. 2004), and
(iv) by altering the target site through 16S RNA mutation
(Ross et al. 1998). Of these, tetracycline efflux proteins and
ribosomal protection proteins are by far the most common
forms of resistance encountered among pathogenic and envi-
ronmental bacteria (Chopra and Roberts 2001). As of July
2007, there were 40 identified tetracycline resistance genes,
designated tet and otr genes. Of these, 25 encode efflux
proteins, 11 encode ribosomal protection proteins, 3 are
tetracycline-degrading enzymes, and 1 has an unknown
mechanism (Roberts 2007). As the trend continues, this list
will surely increase in the future as new resistant isolates are
regularly found around the world.
Efflux proteins
Active tetracycline efflux is the most clinically relevant
and efficient resistance mechanism. Based on a simple yet
highly effective expression system, these proteins can confer
resistance even in the presence of large amounts of tetracy-
cline. Most of the efflux proteins are of gram-negative ori-
gin and are encoded on large conjugative plasmids that
carry other resistance genes (Chopra and Roberts 2001),
which contributes to their rapid spread among bacteria.
These resistance genes encode a membrane-bound efflux
protein belonging to the major facilitator superfamily
(Paulsen et al. 1996). The expression of the gram-negative
efflux proteins is very elegantly regulated by a divergently
positioned transcription repressor gene belonging to the
TetR family, whereas gram-positive efflux genes are regu-
lated by an attenuation mechanism (Poole 2005, 2007). The
best studied efflux system is the gram-negative TetA pro-
tein, which is regulated by the TetR transcriptional re-
pressor. The tetA and tetR genes are divergently positioned,
separated by two identical operator sequences that are 11 bp
apart. In the absence of tetracycline, the TetR protein binds
to the operator sequences as a homodimer and blocks access
to the promoters of both tetA and tetR (Ramos et al. 2005;
Hillen and Berens 1994). When tetracycline in complex
with Mg
2+
is present in the cell, it binds to the TetR homo-
dimer with an association constant of *10
9
(mol/L)
1
,
which is a much tighter binding compared with the associa-
tion constant of *10
6
(mol/L)
1
between tetracycline and
the ribosome (Takahashi et al. 1991; Hinrichs et al. 1994).
The fact that tetracycline binds TetR with a 1000-fold
higher affinity compared with the ribosome ensures that the
required resistance genes are expressed before tetracycline
has a chance to inhibit protein synthesis. When
tetracyclineMg
2+
binds to the TetR homodimer, it induces
a conformation change whereby the two DNA binding do-
mains move apart from 34 to 39 A

and can no longer bind

to the promoter sequences (Hinrichs et al. 1994), leading to
the expression of both TetA and TetR. TetA becomes local-
Fig. 3. Tetracycline resistance mechanisms.
Zakeri and Wright 129
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2008 NRC Canada
ized into the cytoplasmic membrane, where it pumps out
tetracyclineMg
2+
in exchange for a proton in an energy-
dependent manner driven by the proton motive force
(Lomovskaya and Watkins 2001). This leads to rapid and
efficient resistance to high levels of tetracycline, which is
repressed once tetracycline concentrations become suffi-
ciently low.
Ribosomal protection proteins
RPPs represent a class of resistance determinants that are
found among a wide variety of bacteria. They are soluble
cytoplasmic proteins that show significant sequence similar-
ity with the elongation factors and do indeed possess
GTPase activity similar to EF-G and EF-Tu (Burdett 1991).
The best characterized RPPs are Tet(O) and Tet(M), which
function by binding to the ribosome and inducing the
dissociation of the tetracyclineribosome complex in a
GTP-dependent manner (Burdett 1996; Connell et al. 2003).
It has been shown that Tet(O) binds the ribosome near the
A-site in a similar position to EF-G, with which it also
shares structural similarity (Spahn et al. 2001). Although
the RPPs and elongation factors share many similarities,
Burdett has shown that Tet(M) cannot substitute for the
function of EF-G or EF-Tu (Burdett 1996). RPPs confer re-
sistance by freeing the ribosome from tetracycline bound at
the Tet-1 site whereby the drugs are released into the cyto-
sol. Consequently, tetracycline is readily available to bind
the ribosomes at any of the available six sites, which may
explain why RPPs can only provide resistance at low tetra-
cycline concentrations (Pioletti et al. 2001).
Enzymatic inactivation and rRNA mutation
Two alternative means by which bacteria can gain resist-
ance to tetracyclines are through enzymatic inactivation and
16S RNA mutation, both of which are presently of little
clinical significance. The first reported and only character-
ized tetracycline inactivating enzyme is TetX. It was iso-
lated from a plasmid in Bacteroides fragilis, an obligate
anaerobe, but did not confer resistance to its host (Guiney
et al. 1984). Once transferred to E. coli and grown under
aerobic conditions, TetX was able to confer tetracycline re-
sistance (Speer and Salyers 1988; Park and Levy 1988). Re-
cently, we have been able to decipher the molecular
mechanism by which TetX inactivates tetracycline. TetX is
a flavin-dependent monooxygenase that irreversibly hydrox-
ylates tetracycline at position 11a in the presence of
NADPH and O
2
. The product of the reaction is highly unsta-
ble and rapidly undergoes nonenzymatic decomposition at
physiological pH (Yang et al. 2004; Moore et al. 2005). As
mentioned earlier, the hydrophilic side of tetracycline is re-
quired for key hydrogen bonding interactions with the ribo-
some, which is chelated by the substituents on C11 and C12
(Fig. 2a). By adding a hydroxyl moiety at C11a, TetX dis-
rupts the tetracyclineMg
2+
complex and renders the com-
pound inactive (Yang et al. 2004). Interestingly, we have
demonstrated that TetX is capable of inactivating tigecycline
by a similar mechanism (Moore et al. 2005). This is note-
worthy because, as mentioned earlier, tigecycline is a prom-
ising new antibiotic that maintains its bioactivity in the
presence of clinically relevant resistance mechanisms
(Slover et al. 2007), and its inactivation by TetX may have
a profound outcome on its future efficacy. Although TetX is
not currently found amongst pathogenic bacteria, the fact
that it was isolated from a conjugative R plasmid that can
be passed between bacteria through horizontal gene transfer
may be a cause for concern (Guiney et al. 1984; Moore et
al. 2005).
Clinical isolates of tetracycline-resistant cutaneous propio-
nibacteria have been reported. These organisms are associ-
ated with the pathogenesis of acne for which tetracycline
therapy is a common treatment. Analysis of the resistant iso-
lates led to the identification of a single base mutation in the
16S RNA sequence. A mutation of cytosine to guanine at
the equivalent position of base 1058 in E. coli renders tetra-
cycline ineffective (Ross et al. 1998). Since this is a ge-
nomic mutation and is not present on mobile genetic
elements, resistance isolates are confined to a restricted
niche.
Resistance is the Achilles heel of all antibiotics and is
the inevitable outcome of their use. To compete with the
evolution of resistance we must constantly try to discover
or develop new compounds. This requires an in-depth under-
standing of the biosynthetic machinery that makes these
complex natural products, which then might act as a blue-
print for the design of novel compounds.
Tetracycline biosynthesis
Microbes invest large amounts of cellular energy for the
synthesis of an array of secondary metabolites with varying
degrees of complexity, which have been used to treat many
human ailments. Natural products have a wide variety of bi-
ological functions to aid the host organism, including their
highly exploited antimicrobial activity, hormone-like regula-
tion of differentiation, quorum sensing, roles in metal trans-
port, and diffusible siderophores (Challis and Hopwood
2003; Linares et al. 2006). These activities are critical to
the survival of the host organisms in the harsh and competi-
tive environments in which they live. It is therefore impor-
tant to understand how these molecules are made so that we
can take advantage of the efficiency of biological systems to
increase the chemical diversity that is available to us.
The biosynthesis of CTC and OTC, the first two tetracy-
clines discovered, was first studied in the early 1960s
through blocked mutant analysis, substrate feeding
experiments, and precursor analysis (Miller et al. 1964;
McCormick et al. 1965, 1968; McCormick and Jensen
1965, 1969; Barbatschi et al. 1965).
However, it was not until recently that we have come to
better understand the mechanisms by which these two com-
pounds are made and the genetic machinery involved in the
process. The biosynthetic gene clusters that encode CTC
(Ryan 1999; Nakano et al. 2004) and OTC (Zhang et al.
2006) synthesis have been sequenced and have provided an
insight into tetracycline biosynthesis. The genetic structure
and enzymatic mechanisms for the biosynthesis of the two
antibiotics are very similar and therefore only OTC biosyn-
thesis will be discussed henceforth.
Streptomyces rimosus, the OTC producer, has an 8 Mb
linear chromosome with the OTC biosynthetic cluster situ-
ated approximately 600 kb from one end of the chromosome
which contributes to its genetic instability (Pandza et al.
130 Biochem. Cell Biol. Vol. 86, 2008
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2008 NRC Canada
1997). It is composed of 21 open reading frames situated in
between two resistance genes, one encoding an efflux pump
and the other an RPP (Zhang et al. 2006; Chopra and
Roberts 2001). Naturally, the producing organism must en-
code a self-resistance mechanism to protect itself from the
harmful actions of the metabolite. Similarly, other antibi-
otic-resistance genes are also commonly found embedded in
the respective biosynthetic clusters of their substrate rather
than in other parts of the genome. This genetic structure
allows the coregulation of metabolite production and self-
resistance to reduce the energetic burden to cells if the re-
sistance mechanisms are constitutively expressed (Hubbard
and Walsh 2003; Cundliffe 1989). In the case of tetracy-
cline efflux proteins, it has been shown that constitutive
expression of TetA leads to cell toxicity through disruption
of the membrane potential, which eventually causes cell
death (Eckert and Beck 1989). Consequently, tetracycline
biosynthesis requires the coordinated expression of an ef-
flux protein, both for self-defense and to expel the drug
out into the environment as a weapon to combat against
competing microbes. With the recent availability of ge-
nomic information and studies of the origins of these re-
sistance determinants, it is becoming increasingly apparent
that the producing organisms represent a vast reservoir of
resistance genes that can be passed between bacteria of di-
verse genetic backgrounds (Davies 1994; DCosta et al.
2006).
Tetracyclines belong to the polyketide class of natural
products and are produced by type II polyketide synthases
(PKS). Unlike type I PKSs and nonribosomal peptide syn-
thetases (NRPS), which are single mega-enzyme complexes
containing many distinct modules responsible for increasing
the nascent polyketide or peptide chain, respectively, type II
PKSs function as separate subunits (Lai et al. 2006). Conse-
quently, many distinct enzymes perform the tailoring reac-
tions that form the richly decorated polyketide compounds,
and in the process, produce highly unstable intermediates.
The disseminated nature of this mechanism and the chal-
lenges of isolating reaction intermediates has led to many
difficulties in studying this process, which is still poorly
understood (Hertweck et al. 2007). The type II PKS machi-
nery is structured around a minimal PKS complex that is re-
sponsible for producing the nascent polyketide chain. The
minimal PKS comprises two ketosynthase subunits (KS
a
and KS
b
) and an acyl carrier protein (ACP), which are gen-
erally found as three genes clustered together, as within the
OTC biosynthetic cluster (Hertweck et al. 2007). The ACP
acts as an anchor to which the growing nascent polyketide
chain is attached through a thiol moiety. The ACP then
presents the poly-b-keto intermediate to the catalytically ac-
tive site of the minimal PKS, the KS
a
subunit. The KS
a
sub-
unit catalyzes a Claisen-type decarboxylative condensation
reaction between malonyl starter units, leading to C-C bond
formation. This is an iterative reaction that continues for a
predetermined number of cycles as controlled by the KS
b
subunit, otherwise known as the "chain length factor"
(Hertweck et al. 2007). KS
b
shows significant sequence
similarity to KS
a
, except for the presence of a cysteine res-
idue in the active site which leads to a lack of catalytic
activity. However, KS
b
determines the length of the final
polyketide chain by counting the number of Claisen-type
condensations that occur (Hertweck et al. 2007). This is
believed to be the general mechanism that leads to the bio-
synthesis of tetracyclines.
Much work has been done to elucidate the biosynthetic
pathway of OTC production, and recently, Tang and co-
workers have proposed an updated model (Zhang et al.
2006). Polyketide synthases utilize a small set of simple or-
ganic acids that are present in the cell as building blocks for
the synthesis of a wide array of chemical scaffolds (Liou
and Khosla 2003). OTC biosynthesis (Fig. 4) utilizes the
simplest starting unit, malonic acid, which contains three
carbon atoms that are iteratively condensed to produce the
tetracycline scaffold. Although it had been accepted for
some time that malonyl-CoA is the building block for tetra-
cyclines, the nature of the priming unit for ACP had been
the subject of much debate. Tetracyclines possess a carbox-
amido group at C2, which could be introduced into the poly-
ketide either as a malonamate priming unit or as a
downstream tailoring reaction (Moore and Hertweck 2002).
In 1999, Hunter and co-workers shed some light on this
matter by producing an oxyK null S. rimosus mutant
(Petkovic et al. 1999; Moore and Hertweck 2002). The
oxyK gene encodes an aromatase that is responsible for
producing the aromatic ring D of tetracyclines early in the
biosynthetic pathway (Zhang et al. 2006), and disruption of
this function would likely affect more downstream reac-
tions. However, the oxyK null mutant produced four trun-
cated polyketides, all containing the unique carboxamido
group, implying that this moiety must be present at the
start of chain synthesis and that malonamate is the priming
unit (Petkovic et al. 1999; Moore and Hertweck 2002). It
can therefore be rationalized that malonamyl-CoA is ini-
tially loaded onto the minimal PKS, which catalyzes the
decarboxylative addition of eight equivalents of malonyl-
CoA to construct the 19-carbon backbone of OTC (Fig. 4)
(Moore and Hertweck 2002; Zhang et al. 2006). Recently,
the Tang group showed that two aromatase/cyclase
enzymes, OxyK and OxyN, are responsible for closing the
polyketide chain and forming rings D, C, and B in that
order, whereas ring A closes spontaneously in the presence
of the terminal amide via C-1/C-18 aldol condensation
(Zhang et al. 2007). This leads to the production of prete-
tramid, the tetracyclic intermediate that is subsequently
decorated by tailoring enzymes. The C-methyltransferase
OxyF methylates C6, producing 6-methylpretetramid
(Zhang et al. 2007), which is a substrate for downstream
oxygenases. The several oxygenases in the biosynthetic
pathway are responsible for installing the keto-enol func-
tionality on the hydrophilic side of the molecule, which
plays an important role in the interaction between tetracy-
cline and the ribosome while also mediating Mg
2+
chelation (Fig. 2). OxyQ, which is homologous to amido-
transferases, is believed to be the enzyme responsible for
transaminating C4, which is then methylated by OxyT, a
putative SAM dependent N-methyltransferase (Zhang et al.
2006). Although many facets of this biosynthetic mecha-
nism and the reactions therein have been studied, the de-
tailed biochemistry involved still remains a mystery.
Studying the enzymology of the proteins in the OTC bio-
synthetic pathway has proven challenging. Attempts made at
disrupting tailoring enzymes in S. rimosus, which in theory
Zakeri and Wright 131
#
2008 NRC Canada
should not affect the function of the minimal PKS, has led
to the production of novel truncated polyketide molecules.
OxyK catalyzes ring closure of the full length polyketide
chain, implying that it acts upon the polyketide after the
minimal PKS has completed the chain. Yet, its disruption
led to the production of four novel polyketides with shorter
chain length compared with that in OTC (Petkovic et al.
1999). Furthermore, disruption of oxyS, which encodes an
anhydrotetracycline oxygenase that hydroxylates C6 late in
the OTC biosynthetic pathway, also led to the production of
a truncated polyketide (Peric-Concha et al. 2005). These re-
sults imply that the minimal PKS (KS
a
, KS
b
, and ACP) may
not constitute a self-sufficient enzyme complex and that it
may require other enzymes in the OTC biosynthetic pathway
for complete activity (Peric-Concha et al. 2005). The prob-
lems encountered with gene deletions in S. rimosus, com-
pounded by the challenges associated with heterologous
expression of biosynthetic proteins and a lack of availability
of appropriate substrates, has hindered biochemical studies
(Zhang et al. 2007). Therefore, investigators have resorted
to reconstituting parts of the pathway in heterologous Strep-
tomyces hosts while providing the genes under study in shut-
tle vectors (Zhang et al. 2007). These efforts have proven
fruitful and have shed light on many unknown aspects of
the OTC biosynthetic pathway.
Future directions
Antibiotics are one of the most successful classes of drugs
that have ever been used in human therapy. They represent a
25 billion $US annual market, which is expected to steadily
increase (Christoffersen 2006). So why are big pharmaceuti-
cal companies shutting down their antibiotic discovery pro-
grams? Although there has been a rise in multidrug resistant
pathogens and there is a desperate need for new antibiotics,
the rewards are not substantial enough to overcome the
Fig. 4. A summary of oxytetracycline biosynthesis in Streptomyces rimosus. (a) The oxytetracycline biosynthetic gene cluster, as determined
by Zhang et al. (2006). (b) An updated model, proposed by Zhang et al. (2006), of the oxytetracycline biosynthetic pathway.
132 Biochem. Cell Biol. Vol. 86, 2008
#
2008 NRC Canada
financial investments required. During the Golden Age of
antibiotic discovery, most of the low hanging fruit, as
termed by Richard Baltz, were being readily isolated from
soil actinomycetes (Baltz 2005), and since that time, the dis-
covery or production of novel antimicrobial compounds has
steadily declined. Recent approaches, which include combi-
natorial chemical synthesis and subsequent high-throughput
screening of large chemical libraries have helped to identify
novel cellular targets and potential lead compounds, but
have failed to produce new antibiotics (Fernandes 2006).
So what does the future hold for tetracyclines? With a de-
tailed understanding of the chemical interactions of tetracy-
clines with their cellular targets, as well as resistance
determinants, there is now the opportunity to design new
members of this family through molecular modelling. By
modifying the biosynthetic cluster of tetracycline-producing
organisms, such as S. aureofaciens and S. rimosus, we will
be able to isolate tetracycline precursors that are suitable
for chemical modifications while maintaining the bioactive
keto-enol residues on the hydrophilic side of the molecule.
The majority of the semisynthetic tetracyclines that have
been investigated have alterations on C5, C6, C7, C8, and
C9. However, modifications of the dimethylamino group on
C4 have been largely overlooked. This substituent appears to
be in open chemical space when tetracycline is bound to the
ribosome (Fig. 2b), yet it makes key hydrogen bonding in-
teractions when in complex with TetR (Hinrichs et al.
1994). In fact, it has been demonstrated that reversal of the
stereochemistry of the C4 dimethylamino moiety of CTC
leads to a 30-fold reduction in affinity for TetR (Degenkolb
et al. 1991). Currently, we are investigating the potential of
C4-substituted tetracyclines as novel antimicrobials that are
able to maintain their ribosomal binding capabilities while
evading TetR regulated resistance.
An intriguing new function for tetracyclines currently
being investigated is their use in cancer treatment. Matrix
metalloproteinases (MMPs) are zinc-dependent endopepti-
dases that degrade the extracellular matrix. During tumori-
genesis, MMP expression increases relative to that in the
surrounding normal tissue and it is believed that they pro-
mote metastasis by degrading the basement membrane of
blood vessel walls, thereby allowing malignant cells to mi-
grate and enter the blood stream (Hidalgo and Eckhardt
2001). MMPs require a Zn
2+
cofactor to act, and this serves
as the primary target for tetracyclines (Acharya et al. 2004).
Not only do tetracyclines chelate the Zn
2+
ion to inhibit
MMP activity, but it is believed that they also affect MMP
expression and proteolitic activation (Hidalgo and Eckhardt
2001). As a result, many chemically modified tetracyclines
have been developed to investigate their MMP inhibitory ac-
tivity, and some have shown promising results. For example,
the compound COL-3, which possesses a tetracycline scaf-
fold without any substituents on C4C9, has been shown to
be a potent inhibitor of MMPs and has entered clinical trials
(Fig. 5) (Acharya et al. 2004). Thus, tetracyclines are more
than simply antimicrobials.
The unique chemical scaffold of tetracyclines not only al-
lows them to interact with numerous biological targets, but
also makes them amenable to, and tolerant of, chemical
modifications. The available genomic information and an
understanding of the chemical biology of tetracyclines pro-
vides us with the necessary tools to rationally design semi-
synthetic compounds to combat emerging multidrug
resistant pathogens. The broad-spectrum antibiotic activity
of tetracyclines makes them a vital weapon against infec-
tious disease and, as evidenced by the development of tige-
cycline, they remain important scaffolds for new chemists.
Acknowledgement
Tetracycline research in our laboratory is funded by the
Natural Sciences and Engineering Research Council of Can-
ada.
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