Microbiology 1

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Bio 351- Homework 1 (10 points)

DUE TUESDAY JANUARY 16th at 11PM on Blackboard. ONLY Word documents accepted.

Go to pages 1-5 of the article: Wall Teichoic Acid Function, Biosynthesis, and Inhibition to answer questions 1-3 below (30 words maximum per answer):

1. What are the differences between cell wall associated teichoic acids (WTA) and lipoteichoic acid (LTA) (2 points)? WTA and LTA are modified by D-alanylation, a process that incorporates D-alanine into teichoic acids. How is D-alanylation beneficial for gram-positive bacteria (2 points)?

2. How does cation-binding influence the stability of a gram-positive surface (cell wall)? (2 points)

3. What roles are teichoic acids involved in that contribute to the stability of a gram-positive cell wall? (2 points)

Correct the Statement (1 point each): The statements from each question below are false. Correct the statement by crossing out a word/words. The statement must be correct, not partially correct. Please do not rewrite your corrected statement. You must cross out the word on this document, and manually correct each statement.

4. Lipoteichoic acids are seen within the plasma membrane of E. coli.

5. Lipopolysaccharides are molecules seen within the periplasm of gram positive bacteria.

Wall Teichoic Acid Function, Biosynthesis, and Inhibition

Jonathan G. Swoboda, Jennifer Campbell, Timothy C. Meredith, and Suzanne Walker
Department of Microbiology and Molecular Genetics Harvard Medical School, 200 Longwood
Avenue Armenise 633, Boston, MA 02115 (USA) Fax: (+ 1) 617-738-7664

Keywords
antibiotics; biosynthesis; conditionally essential enzymes; Gram-positive bacteria; wall teichoic acid
(WTA)

Introduction
One of the major differences between Gram-negative and Gram-positive organisms is the
presence or absence of an outer membrane (Figure 1). In Gram-negative organisms, the outer
membrane protects the organism from the environment. It filters out toxic molecules and
establishes a compartment, the periplasm, which retains extracytoplasmic enzymes required
for cell-wall growth and degradation. It also serves as a scaffold to which proteins and
polysaccharides that mediate interactions between the organism and its environment are
anchored.[1] In addition, in ways that are not completely understood, the outer membrane
functions along with a thin layer of peptidoglycan to help stabilize the inner membrane so that
it can withstand the high osmotic pressures within the cell.[2]

Gram-positive organisms, in contrast, lack an outer membrane and a distinct periplasm (Figure
1). The peptidoglycan layers are consequently very thick compared to those in Gram-negative
organisms.[4] These thick layers of peptidoglycan stabilize the cell membrane and also provide
many sites to which other molecules can be attached. Gram-positive peptidoglycan is heavily
modified with carbohydrate-based anionic polymers that play an important role in membrane
integrity.[5] These anionic polymers appear to perform some of the same functions as the outer
membrane: they influence membrane permeability, mediate extracellular interactions, provide
additional stability to the plasma membrane, and, along with peptidoglycan, act as scaffolds
for extracytoplasmic enzymes required for cell-wall growth and degradation.

A major class of these cell surface glycopolymers are the teichoic acids (TAs), which are
phosphate-rich molecules found in a wide range of Gram-positive bacteria, pathogens and
nonpathogens alike. There are two types of TAs: the lipo-TAs (LTAs), which are anchored to
the plasma membrane and extend from the cell surface into the peptidoglycan layer;[6] and the
wall TAs (WTAs), which are covalently attached to peptidoglycan and extend through and
beyond the cell wall (Figure 1).[7] Together, LTAs and WTAs create what has been aptly
described as a “continuum of negative charge” that extends from the bacterial cell surface
beyond the outermost layers of peptidoglycan.[5] Neuhaus and Baddiley comprehensively
reviewed both LTAs and WTAs in 2003.[5] Since then, however, new functions for WTAs in
pathogenesis have been uncovered and it has been suggested that the biosynthetic enzymes
that make these polymers are targets for novel antibacterial agents.[8,9] Indeed, the first WTA-

© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
suzanne_walker@hms.harvard.edu.

NIH Public Access
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Chembiochem. 2010 January 4; 11(1): 35–45. doi:10.1002/cbic.200900557.

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active antibiotic has just been reported.[10] This review will focus primarily on recent
developments in the study of WTAs in Bacillus subtilis and Staphylococcus aureus, and will
include a discussion of strategies for the discovery of WTA inhibitors and prospects for these
inhibitors as antibiotics.

Wall Teichoic Acid Structure
WTAs are anionic glycopolymers that are covalently attached to peptidoglycan via a
phosphodiester linkage to the C6 hydroxyl of the N-acetyl muramic acid sugars.[5] They can
account for as much as 60 % of the total cell wall mass in Gram-positive organisms. The
chemical structures of WTAs vary among organisms, as described in detail by Neuhaus and
Baddiley,[5] but the most common structures are composed of a ManNAc(β1→4)GlcNAc
disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc
residue (the “linkage unit”) followed by a much longer chain of glycerol- or ribitol phosphate
repeats (the “main chain”; Figure 2).[11–18] B. subtilis, the Gram-positive model organism,
makes poly(glycerol phosphate) or poly(ribitol phosphate) WTAs depending on the strain,
[19] while S. aureus strains primarily make poly(ribitol phosphate) WTAs.[20–23] The
hydroxyls on the glycerol- or ribitol phosphate repeats are tailored with cationic D-alanine esters
and monosaccharides, such as glucose or N-acetylglucosamine.[24,25] The presence of WTAs
and the particular tailoring modifications that are found on them have profound effects on the
physiology of Gram-positive organisms, and impact everything from cation homeostasis to
antibiotic susceptibility to survival in a host.

Functions of Teichoic Acids in Bacterial Physiology
The functions of TAs in bacterial physiology are incompletely understood, but evidence for
their importance is overwhelming. B. subtilis and S. aureus mutants deficient in LTA
biosynthesis can be obtained but only if grown under a narrow range of conditions; they are
temperature sensitive and exhibit severe growth defects.[26,27] Mutants deficient in WTA
biosynthesis are also compromised and manifest increased sensitivity to temperature and
certain buffer components, including citrate; they also tend to aggregate in culture.[26–31] In
addition, B. subtilis strains that do not express WTAs show profound morphological
aberrations. Bacterial strains in which both LTA and WTA expression are prevented are not
viable, an observation suggesting that these polymers have overlapping functions and can
partially compensate for one another.[26,27] Indeed, this might be expected for some functions
since both polymers contain phosphate-linked repeat units with similar tailoring modifications.
One of the tailoring modifications, D-alanylation, is accomplished by the same machinery, so
there is even some overlap in the biosynthetic pathways. This fact makes dissecting the
functions of the individual anionic glycopolymers difficult, but is consistent with the idea that
LTAs and WTAs are partially redundant. Some of the functions attributed to WTAs are
described in the following paragraphs. LTAs are beyond the scope of this review, but will be
mentioned in cases where it is relevant to the discussion of WTAs. Morath et al. and Rahman
et al. have each written recent reviews on LTA structure and biosynthesis.[6,32]

Cation binding functions
WTAs form a dense network of negative charges on Gram-positive cell surfaces. To alleviate
the resulting electrostatic repulsive interactions between neighboring phosphates, TAs bind
cationic groups, including mono- and divalent metal cations. Networks of WTA-coordinated
cations affect the overall structure of the polymers, and this in turn influences the porosity and
rigidity of the cell envelope. WTAs are proposed to be important for cation homeostasis in
Gram-positive organisms,[33,34] and provide a reservoir of ions close to the cell surface that
might be required for enzyme activity. In addition, the gradient of ions could in some way
mitigate the osmotic pressure change between the inside and outside of the cell. The amount

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of bound cations can be modulated by D-alanylation, a tailoring modification that introduces
positively charged amines.[35] WTAs that lack D-alanyl esters can bind up to 60 % more
Mg2+ ions than analogous polymers that contain this modification.[36] The importance of
cation binding is highlighted by the observation that B. subtilis strains up-regulate their
production of TAs in the presence of low Mg2+ concentrations, and produce other negatively
charged polymers (teichuronic acid) in the presence of limiting phosphate concentrations.
[37] Recent structural studies have been focused on elucidating modes of cation binding by
WTA polymer phosphate groups, and researchers have suggested that a clear understanding
of the three-dimensional structure of WTAs and their bound cation groups might provide
insights that facilitate the design of novel antimicrobials.[38]

Scaffolding roles
In addition to providing binding sites for cations, WTAs serve as scaffolds or receptors for a
wide range of other molecules. In S. aureus, for example, they function as receptors that are
required for phage infection.[39] Depending on their tailoring modifications (see below) they
might also promote adhesion by lytic enzymes produced by neutrophils.[40] They are
additionally thought to serve as scaffolds for endogenously produced cell wall hydrolases
(autolysins) involved in cell growth and division.[41] In general, the molecular interactions
between WTAs and other biomolecules are not well understood but could provide crucial
insights into cell envelope function.

Tailoring modification-dependent functions
The main chain hydroxyl groups on both glycerol- and ribitol phosphate WTA polymers are
subject to further derivatization by tailoring enzymes (Figure 2). There are two classes of
tailoring enzymes: those that catalyze the addition of D-alanyl esters, and those that append
glycosyl groups. The extent to which these modifications occur on the TA polymers is strain
dependent and can also be affected by environmental conditions. Efforts have been made to
understand the role(s) of these modifications in bacterial physiology, and some of these studies
are highlighted below.

The D-alanylation tailoring modification has been more extensively investigated than
glycosylation and is far better understood at this point. Perego et al. were the first to characterize
the genetic pathway responsible for this modification (dlt operon) in B. subtilis.[42] Briefly,
the biosynthetic pathway begins intracellularly with the activation of D-alanine to its
corresponding aminoacyl adenylate by DltA. This molecule is then covalently attached, as a
thioester, to a cofactor bound to the D-Ala carrier protein, DltC. Although the precise roles of
DltB and DltD have not been confirmed, it is believed that they facilitate the transport of DltC
through the membrane and the incorporation of D-Ala onto both LTAs and WTAs.[43] It has
been found that D-alanylation is affected by several factors, including growth media, pH and
temperature.[5] The attachment of D-alanyl esters to the hydroxyls on TAs alters the net charge
of the polymer by adding positively charged amines. This modification reduces the electrostatic
repulsion between neighboring TA chains and possibly facilitates stabilizing ion-pair
formation between the cationic esters and the anionic phosphate groups.[38]

The D-alanine modification modulates interactions between the cell envelope and the
environment and has been implicated in many of the known scaffolding/receptor functions of
WTAs.[5,44] For example, it has been shown that the absence of D-alanyl esters on the TA
polymers increases susceptibility to cationic antimicrobial peptides, possibly by increasing the
negative charge density on the cell surface.[45,46] Removing the alanine residues also
increases bacterial sensitivity to glycopeptide antibiotics and to the lytic activity of enzymes
produced by neutrophils during host infection.[40,41] In contrast, the activity of autolytic
enzymes is decreased, suggesting a role for TAs in scaffolding and/or activating bacterial

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enzymes involved in the processes of cell-wall synthesis and degradation.[41] Removal of D-
alanyl esters from TAs has also been shown to attenuate the binding of S. aureus to artificial
surfaces as well as host tissue. A recent study has illustrated the importance of the charge
balance of WTAs in adhesion to artificial surfaces, such as glass and polystyrene.[44]

Since D-alanylation promotes better adhesion to host tissue and confers some resistance to lytic
enzymes produced by the host, mutant strains lacking this modification have been studied in
animal infection models. For example, in a mouse tissue cage infection model, bacterial strains
lacking D-alanylation were more susceptible to Toll-like receptor 2-dependent host defenses;
[46] in a septicemia model, such strains were attenuated in their ability to establish an infection,
possibly because they were more readily killed by neutrophils.[40] Based on these and other
studies, it was proposed that the D-alanine modification is a putative target for novel
antimicrobials that function by attenuating virulence. In 2005, May et al. reported the synthesis
and evaluation of a nonhydrolysable analogue of D-Ala aminoacyl adenylate as the first
designed inhibitor of DltA, the enzyme that activates D-Ala. The compound enhanced the
activity of vancomycin against B. subtilis.[43] This result is consistent with inhibition of DltA,
and supports the idea that small molecules that interfere with D-alanylation might provide a
novel strategy for antimicrobials.

Glycosylation is a ubiquitous tailoring modification of WTAs but its functions are not well
understood. Glucose is commonly added to the WTA polymers in B. subtilis, whereas N-acetyl
glucosamine (GlcNAc) is added in S. aureus (Figure 2).[5] Depending on the bacterial strain,
the stereochemistry of the glycosidic linkage may be β-, α-, or a mixture of the two anomers.
All sequenced B. subtilis and S. aureus strains contain one or more putative glycosyltransferase
genes clustered with the WTA biosynthetic genes (Figure 3). For example, B. subtilis 168
contains a gene for a putative retaining glycosyltransferase that might add a-Glu to the glycerol
phosphate polymers. S. aureus strains contain two genes encoding putative inverting
glycosyltransferases that might transfer β-GlcNAc to the poly(ribitol phosphate) polymers.
Although some S. aureus strains have been shown to contain α-glycosidically linked WTAs,
there are no genes yet identified for any glycosyltransferases that can carry out this tailoring
modification. Furthermore, no studies have confirmed the enzymatic functions of any of the
putative WTA glycosyltransferases or have explored the effects of preventing WTA
glycosylation on bacterial cell growth, division, intercellular interactions, or pathogenesis. In
fact, as far as we know there is only one piece of data pertaining to the functions of WTA
glycosyltransferases in the literature: a transposon mutant in a putative glycosyltransferase in
the S. aureus strain Newman showed attenuated virulence in a nematode killing assay,
suggesting that glycosylation might play a role in pathogenesis in S. aureus.[47] If
glycoslyation proves important for bacterial pathogenesis, the glycosyltransferase tailoring
enzymes, like the enzymes involved in D-alanylation (see above) would be possible targets for
antimicrobials.

Roles in cell elongation and division
Recent studies have implicated LTAs and WTAs in cell growth, division, and morphogenesis.
In the rod-shaped organism B. subtilis, TAs have been shown to play distinct roles in bacterial
morphogenesis. Preventing WTA expression results in the production of round, severely
defective progeny, while preventing LTA biosynthesis causes major defects in septum
formation and cell separation.[27,49] It is known that there are separate multiprotein complexes
involved in septation and elongation in B. subtilis, and Errington and co-workers have
suggested (based on localization studies using fluorescently tagged enzymes) that the WTA
biosynthetic enzymes associate with the machinery involved in elongation, while the LTA
enzymes might associate with machinery involved in septation and cell division.[27,50] It was
suggested that the spatial distribution of these two anionic glycopolymers determines their

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specific functions. Defects in S. aureus upon deletion of WTAs are less pronounced than in
B. subtilis, and no specific roles in cell growth and division for WTAs in this organism have
been proposed. However, Oku et al. recently reported that S. aureus strains devoid of LTAs
show major defects in septal formation and cell separation and grow only under a restricted
range of conditions, including reduced temperatures.[26]

Functions in biofilm formation and host tissue adhesion
As major components of the cell envelope, WTAs influence the interactions of bacterial cells
with their environment in many ways. We have already mentioned that S. aureus mutants
lacking WTAs show reduced initial adherence to artificial surfaces, including glass and
polystyrene;[44] they are also impaired in their ability to form biofilms. It has been shown that
WTA null mutants that are impaired in biofilm formation do not have a reduced production of
the exopolysaccharide poly-N-acetylglucosamine (PNAG), which has been identified as an
important factor for biofilm formation.[30] This finding highlights the independent role that
WTAs play in biofilm formation.

S. aureus WTAs are also required for adhesion to host tissue. Peschel and co-workers have
shown that S. aureus strains that do not express WTAs are severely impaired in their ability to
adhere to nasal epithelial cells and are unable to colonize the nasal passages of cotton rats.[8]
They have also shown that WTA-null mutants cannot colonize endothelial tissues derived from
kidney and spleen.[9] The D-alanylation machinery was not impaired in these strains, and D-
alanylation could still have occurred on LTAs; therefore, these results implicate WTAs as
independent factors involved in cell adhesion. Since WTAs are required for host infection and
play important roles in biofilm formation, it was suggested that they are virulence factors, that
is, factors required for the establishment and spread of infection in a host. Therefore, the
enzymes involved in WTA biosynthesis were suggested to be targets for novel antimicrobials
that impede host colonization by S. aureus.[7]

Biosynthesis of Wall Teichoic Acids
Poly(glycerol phosphate) WTA biosynthesis in B. subtilis 168

The pathway for WTA biosynthesis was first characterized in B. subtilis 168, which makes
poly(glycerol phosphate) WTAs (Figure 4 A).[51] The genes involved in the synthesis of these
WTAs are known as tag genes (for teichoic acid glycerol). The pathway starts in the cytoplasm
with the transfer of GlcNAc phosphate to an undecaprenyl phosphate (also known as
bactoprenyl phosphate) carrier anchored in the bacterial membrane. The enzyme that catalyzes
this reaction, TagO, is reversible and is related to a large family of phosphosugar transferases
that includes the first enzyme in the dolichol pathway for N-linked glycosylation in eukaryotes,
GPT, as well as MraY, an essential bacterial enzyme involved in peptidoglycan biosynthesis.
[52,53] Following formation of the GlcNAc-pp-lipid by TagO, an N-acetylmannosaminyl
transferase, TagA, transfers ManNAc from UDP-ManNAc to the C4 hydroxyl of the GlcNAc
residue to form a β-linked disaccharide, which is the substrate for the next enzyme in the
pathway, TagB.[54,55] TagB is a glycerophosphate transferase that transfers a single
phosphoglycerol unit from CDP-glycerol to the C4 hydroxyl of ManNAc to complete the
synthesis of the linkage unit (Figure 2).[54,56] The next enzyme in the B. subtilis 168 pathway,
TagF, is a polymerizing cytidylyl transferase that attaches 35 or more glycerol phosphates to
the linkage unit to form the anionic polymer.[57–59] The catalytic domains of TagB and TagF
share significant sequence identity and belong to a group of phosphotransferases that are
apparently unique to WTA biosynthesis. Other members of this family include TarB, F, K, and
L (see below). Once assembled, the lipid-linked WTA polymer is putatively modified by a
glycosyltransferase (TagE) and then exported to the external surface of the bacterial membrane
by a two-component ABC (ATP binding cassette) transporter, TagGH.[60] The polymer is

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coupled to peptidoglycan through the anomeric phosphate of the GlcNAc residue. The
transferase responsible for carrying out this reaction has not been identified. D-Alanine ester
formation occurs outside of the cell as described above.[5]

Poly(ribitol phosphate) WTA biosynthesis in B. subtilis W23
B. subtilis W23 makes a poly(ribitol phosphate) WTA rather than a poly(glycerol phosphate)
WTA (Figure 2). A pathway for the biosynthesis of B. subtilis W23 WTA was proposed by
Lazarevic et al., who designated the genes involved as tar genes (for teichoic acid ribitol).
[19] The first three steps of the proposed pathway, mediated by TarO, TarA, and TarB are
identical to those in B. subtilis 168, but the pathways then diverge (Figure 4 B). The TagF
homologue in B. subtilis W23, TarF, functions not as a polymerase but as a primase, and adds
one additional glycerol phosphate unit to the 168-type linkage unit. The catalytic domains of
TagF, which is a polymerase, and TarF, which is a primase, share significant sequence identity
and the structural features in each enzyme that determine whether one or many glycerol
phosphate units is transferred to the linkage unit have not been identified. Once the W23 linkage
unit is complete, the poly(ribitol phosphate) main chain is assembled. Lazarevic et al. proposed
that the assembly of this poly(ribitol phosphate) chain requires two enzymes: TarK, which
transfers a single ribitol phosphate residue to the linkage unit, and TarL, which carries out the
polymerization of the ribitol phosphate chain.[19] TarK and TarL in B. subtilis W23 were thus
suggested to function as a primase/polymerase pair, analogous to the primase/polymerase pair
(TagB/TagF) that assembles the poly(glycerol phosphate) chain in strain 168. Meredith et al.
used a genetic approach to verify that tarK and tarL from B. subtilis W23 are both required for
the assembly of poly(ribitol phosphate) WTAs;[61] this is consistent with the proposed
primase/polymerase model for biosynthesis. Pereira et al. subsequently confirmed this finding.
[62] Once the poly(ribitol phosphate) WTA polymer is assembled, the remaining steps are
thought to be similar to those in strain 168. That is, the WTA polymer is glycosylated,
transported through the bacterial membrane by a two-component transporter, TarGH, attached
to peptidoglycan by an unidentified transferase, and esterified with D-alanine residues.

Poly(ribitol phosphate) WTA biosynthesis in S. aureus
Like B. subtilis W23, S. aureus also makes a ribitol phosphate WTA polymer. Except for the
length of the polymer chain and the nature of the appended sugar residues, the structures of
the poly(ribitol phosphate) WTAs are thought to be the same in B. subtilis W23 and S.
aureus (Figure 2). The assembly of the linkage unit in S. aureus is identical to its synthesis in
B. subtilis (TarO, TarA, TarB, TarF catalyze the same reactions), but the main chain is
assembled not by a primase/polymerase pair, but by one of two bifunctional poly(ribitol
phosphate) primase/polymerases (currently designated TarK and TarL although their functions
are different from TarK/TarL in B. subtilis; Figure 4 C). It has been proposed that S. aureus
TarL makes a primary WTA polymer (L-WTA) while S. aureus TarK makes a secondary WTA
polymer (K-WTA).[61] As outlined in the following section, however, there are still a number
of questions about the cellular roles of the two bifunctional poly(ribitol phosphate) polymerases
in S. aureus. Once the ribitol phosphate polymer is completed in the cytoplasm, glycosylation
occurs and the polymer is flipped to the external surface of the membrane by an ABC-dependent
transporter complex (TarGH) before ligation to the cell wall by unidentified enzyme(s) and D-
alanylation.

Gene Cluster Duplication in S. aureus
As noted in the previous section, S. aureus contains two bifunctional poly(ribitol phosphate)
polymerases that have similar enzymatic functions rather than a pair of enzymes containing
separate primase and polymerase activities. Qian et al. were the first to note that S. aureus may
differ from B. subtilis in how it accomplishes poly(ribitol phosphate) polymerization. In a

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genomic analysis of six sequenced S. aureus strains (Figure 3)[48] these authors noted that all
of the strains contained an apparent duplication of the region of the chromosome containing
the putative ribitol phosphate polymerase gene and the two genes involved in the synthesis of
its CDP-ribitol substrate (tarI,J,L). These two similar gene clusters have since been designated
in the literature as tarI,J,L and tarI′,J′,K. The tarK gene is highly homologous to the tarL gene,
which suggests it might have the same enzymatic function. Walker and co-workers provided
the first experimental evidence for a unique S. aureus-specific poly(ribitol phosphate) WTA
biosynthetic pathway. By utilizing an approach previously developed to study peptidoglycan
biosynthesis[63,64] and later applied to validate part of the B. subtilis WTA pathway,[54] the
Walker group reconstituted the biosynthesis of S. aureus poly(ribitol phosphate) WTA in vitro.
[65] Through the use of synthetic substrates, it was shown that S. aureus TarL is, in fact, a
bifunctional enzyme that combines both primase and polymerase activities (Figure 4).[65]
They demonstrated that a dedicated ribitol phosphate primase was not required for WTA
polymer synthesis in S. aureus as it is in B. subtilis.

The in vitro studies of WTA biosynthesis established the enzymatic function of TarL in S.
aureus, but did not answer the question of why S. aureus contains an additional gene, tarK,
that is homologous to tarL. Meredith et al. and Pereira et al. used genetics to probe the cellular
roles of tarK and tarL.[61,62] Under certain conditions, it was shown that tarK can compensate
for the loss of tarL, and both groups have concluded that TarK, like TarL, is a bifunctional
enzyme that combines ribitol phosphate primase and polymerase activities. However, Meredith
et al. and Pereira et al. have proposed alternative explanations for the cellular roles of tarK and
tarL. Pereira et al. have suggested that tarK is a redundant gene resulting from duplication, and
have argued that its function is decaying. Meredith et al. reached a different conclusion based
on an in-depth analysis of tarK and tarL expression in cells. Analysis of extracted WTAs from
strains that produce only TarL or TarK showed that these two enzymes produce
electrophoretically distinct poly(ribitol phosphate) WTAs, designated L-WTA and K-WTA
(Figure 5 A). K-WTA is significantly shorter than L-WTA and is presumed, based on PAGE
banding patterns, to contain subtle differences in composition. Furthermore, K-WTA
biosynthesis is negatively regulated (directly or indirectly) by the agr (accessory gene
regulator) quorum sensing system.[61] Since tarK expression can cause the polymer to shorten
by up to 50 %,[61] it was suggested that regulation of tarK by agr allows S. aureus to
dynamically control WTA chain length as a function of cell density. WTA polymer length
might affect exposure of surface adhesins, and it was proposed that dynamic regulation of WTA
polymer length allows S. aureus to cycle between a pro-adhesion state and a low-adhesion
state, perhaps to promote adhesion and dissemination at appropriate times during the infection
process (Figure 5 B). Determination of the exact structures of K-WTA and L-WTA and their
potential roles in virulence remain to be addressed.

Disagreement about the functions of tarK and tarL extends to other genes within the two tarI
′J′K/IJL clusters. It has been reported that tarI′ and tarI are both nonessential,[66] that only
tarI is essential,[67] or that tarI is only essential under a certain set of growth conditions in
vitro but is nonessential in an in vivo infection model.[68] Furthermore, in S. aureus Newman,
seven viable transposants were isolated within tarI′J′K but none was isolated in tarIJL,[47]
suggesting that the gene duplications are not redundant. Recently, Chaudhuri et al. have
reported that tarI, tarJ, and tarL are essential in S. aureus.[69] These discrepancies can be
collectively resolved by suggesting that there are differences in tarI′J′K expression, which
depend on culture conditions and strain backgrounds. The fact that all fourteen sequenced S.
aureus strains retain both tarI′J′K and tarIJL intact, argues against simple functional
redundancy and suggests a selective pressure for maintaining both clusters.

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Characterization of the Wall Teichoic Acid Biosynthetic Genes in S. aureus
WTA biosynthesis exhibits a mixed gene dispensability pattern

It was first observed in the 1960s that WTAs are not essential for the survival of S. aureus in
vitro,[39] and a number of studies on genetically uncharacterized WTA null mutants were
reported in subsequent years.[70–74] In 2004, Peschel and co-workers characterized a defined
WTA-null S. aureus strain lacking tarO, the first gene in the WTA biosynthetic pathway.[8]
This ∆tarO strain was reported to have a similar in vitro growth rate to the wild-type strain,
but was greatly impaired in its ability to adhere to epithelial and endothelial tissues. The
adhesion-impaired mutant was unable to colonize nasal passages, leading to the suggestion
that WTAs might be virulence factors since they are required for host infection. Brown and
co-workers subsequently reported that tarA, like tarO, can also be deleted.[75] The ∆tarA
WTA-null strains are viable in vitro and are phenotypically identical to the ∆tarO strains;
however, many of the genes downstream of tarA in the S. aureus WTA pathway (depicted in
red in Figure 6) cannot be deleted unless tarO (or tarA) is deleted first.[67] These downstream
genes are “conditionally essential”: that is, they are required for viability in a strain background
containing a functional WTA pathway, but are not required in a WTA-null background. This
mixed gene dispensability pattern implies that blocking late-acting WTA biosynthetic enzymes
after flux into the pathway has been initiated is deleterious to bacterial growth.

Possible explanations for conditional essentiality
The mixed gene dispensability pattern observed for WTA biosynthesis has also been reported
for several other nonessential biosynthetic pathways in which a cell surface glycopolymer is
assembled on an undecaprenyl phosphate carrier lipid.[76–80] Such pathways exist in virtually
all bacteria. For example, they are involved in the synthesis of O-antigens, capsular
polysaccharides, and exopolysaccharides. In pathogenic bacteria, these cell surface polymers,
like WTAs, play roles in virulence. Two explanations have generally been considered for the
apparent toxicity caused by blocking late steps in these pathways. One explanation attributes
toxicity to depletion of undecaprenyl phosphate-linked peptidoglycan precursors and the
resulting effects on peptidoglycan biosynthesis. Undecaprenyl phosphate is used as the carrier
lipid in the peptidoglycan biosynthetic pathway; however, only small amounts of this carrier
lipid are produced and the cell’s capacity to increase these levels is limited. Therefore, any
metabolic block that leads to accumulation or sequestration of undecaprenyl phosphate-linked
intermediates is potentially harmful to cells. Indeed, a number of cell-wall active antibiotics,
including bacitracin, vancomycin, and ramoplanin, function by sequestering peptidoglycan
precursors.[81–83] An alternative explanation attributes the observed toxicity upon blocking
nonessential bactoprenol-dependent pathways to an accumulation of bactoprenol-linked
intermediates that are somehow directly harmful to cells.[84] Evidence for and against both
mechanisms has been presented, but a consensus has not yet been reached. Since these
possibilities are not mutually exclusive, it is possible that both play a role.

Brown and co-workers have proposed that peptidoglycan substrate depletion is the mechanism
for toxicity when WTA biosynthesis is blocked in B. subtilis.[85] Microarray analysis was
used to identify genes up-regulated in B. subtilis upon depletion of TagD, the
cytidylyltransferase that provides activated glycerol phosphate for poly(glycerol phosphate)
synthesis. The promoters for ten highly up-regulated genes were then fused to the lux operon
and the luminescence signal upon tag gene depletion was evaluated. One of the promoters,
PywaC, gave a particularly robust luminescent signal when WTA biosynthesis was disrupted at
a late step. The PywaC reporter was activated by cell-wall active antibiotics that sequester
peptidoglycan precursors as well as by depletion of genes involved in undecaprenol
biosynthesis. It was not activated by depletion of tagO. Since the PywaC reporter strain
responded to perturbations known to affect pool levels of bactoprenol-linked peptidoglycan

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intermediates and to late-acting tag gene depletion, it was suggested that blocking late-acting
WTA enzymes is toxic because it affects levels of bactoprenol-linked peptidoglycan substrates.
Brown and co-workers have speculated that their reporter assay can be used in a high-
throughput screen to identify compounds that target either the cell wall or wall teichoic acid
biosynthetic pathways.

Inhibitors of Wall Teichoic Acid Biosynthesis
Methicillin-resistant S. aureus infections have become a major problem in the United States,
recently surpassing HIV/AIDS as a cause of death. Although there are still a handful of effective
anti-MRSA antibiotics, clinical resistance is inevitable and, indeed, has already been observed
for the most recently introduced antibiotics.[86] Thus, an urgent need exists for the exploration
of new strategies to battle resistant S. aureus infections.

The WTA biosynthetic pathway has been speculated to be an antibiotic target for many years,
but only one specific inhibitor has recently been reported. The mixed gene dispensability
pattern implies that there are two distinct types of possible antimicrobial targets within the
pathway: antivirulence targets (TarO and TarA; depicted in green in Figure 6) and antibiotic
targets (the conditionally essential downstream enzymes; depicted in red). Small molecule
inhibitors of the former are expected to impede colonization and the spread of infection, while
inhibitors of the latter have been shown to prevent bacterial growth (see below). The known
inhibitors of WTA biosynthesis are described below.

Inhibitors of antivirulence targets in the WTA pathway
Peschel and co-workers were the first to suggest that WTA biosynthesis is an antivirulence
target in S. aureus.[8] This possibility has attracted considerable attention because it is
speculated that resistance to nonessential targets involved in pathogenicity (virulence factor
targets) will not develop as readily as it does to more traditional antibiotic targets.[87,88] May
et al. have reported a small molecule that inhibits the D-alanine tailoring modification in both
LTAs and WTAs, and the activity of this compound in preliminary studies supports the
possibility that inhibiting D-alanylation could attenuate the virulence of pathogenic organisms.
[43] In addition to this compound, there is a very potent natural product inhibitor of WTA
biosynthesis, the uridine-containing antibiotic tunicamyin (Figure 7).[72,89] Tunicamycin is
a promiscuous inhibitor of the large family of enzymes that couple sugar phosphates to
membrane-embedded lipid phosphates.[52] Its well-known antibacterial activity derives from
its ability to inhibit MraY, an essential phosphosugar transferase in the peptidoglycan
biosynthetic pathway; however, tunicamycin also inhibits TarO.[10,52] In fact, tunicamycin
is selective for TarO over MraY by a factor of at least 100. Its selectivity for TarO makes it a
useful tool for shutting off WTA expression in vitro without affecting bacterial growth rates.
Unfortunately, it cannot be used in animals to assess whether inhibiting TarO is a viable strategy
for treating S. aureus infections because it is toxic to eukaryotes. It inhibits an essential
eukaryotic phosphosugar transferase involved in the dolichol pathway for N-linked
glycosylation (GPT), which catalyzes the same chemical transformation as TarO. Nontoxic,
selective inhibitors of TarO (or TarA) remain to be discovered.

Inhibitors of antibiotic targets in the WTA pathway
The first inhibitor of a putative antibiotic target in the WTA biosynthetic pathway was recently
reported by Swoboda et al.[10] It was discovered by using a general cell-based screening
approach that exploits the conditional essentiality of the late-acting enzymes (Figure 8).[10]
The screening strategy developed to discover WTA inhibitors is applicable, in principle, to any
nonessential biosynthetic pathway containing conditionally essential genes. It involves
screening a compound library against a pair of bacterial strains, one a wild-type strain and the

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other a null mutant that does not express the polymer of interest (e.g., WTAs). Compounds
that inhibit the growth of the polymer-expressing wild-type strain, but not of the mutant, are
expected to target the conditionally essential enzymes in the desired biosynthetic pathway.
Screening paired strains ensures target specificity while eliminating compounds that inhibit
essential cellular processes or are toxic for other reasons (Figure 8). Since antibiotic discovery
is challenging, a cell-based screen that ensures cellular activity is critical, but designing a screen
to report on a particular pathway is typically difficult. The reported strategy combines two
important features that are not often found together in high-throughput screens: it is both cell-
based and pathway specific.

Swoboda et al. used the paired strain screening strategy to identify WTA inhibitors with
antibiotic activity.[10] The growth of S. aureus RN4220 and the corresponding ∆tarO strain
were monitored in the presence of a library of 55 000 small molecules. Three inhibitors were
found to inhibit the wild-type strain without affecting the mutant. The most active of the three
compounds (1835F03; Figure 7) was found to have a minimum inhibitory concentration of 1–
2 µg mL–1 (2.5–5 µM) against all S. aureus strains examined, including clinical MSSA and
MRSA isolates. A comprehensive set of genetic and biochemical experiments have shown that
the target of the compound is TarG, the transmembrane component of the dedicated, two-
component ABC transporter that exports WTAs from the cytoplasm to the cell surface.

The discovery of a small molecule that inhibits a late-acting step in WTA biosynthesis and has
growth inhibitory activity validates the WTA pathway as a possible antibacterial target, but
the efficacy of this antibacterial strategy has yet to be determined. Resistance to the reported
WTA inhibitor occurs at a high frequency in vitro (1 in 106) and two classes of resistant mutants
have been identified. One class involves mutations in the target (TarG), a common theme for
antibiotics. The other mutants contain changes in the tarO or tarA genes, which abolish WTA
expression. The observation that this latter class of mutations occurs frequently is perhaps not
surprising, as the pathway is not essential for growth in vitro. Under ordinary circumstances,
obtaining a high frequency of resistant mutants in vitro would suggest that a particular pathway
is not a viable antimicrobial target. However, the WTA biosynthetic pathway presents an
unusual and previously unexplored paradigm. A large percentage of the resistant mutants do
not express WTAs but, because Peschel and co-workers have reported that S. aureus strains
lacking WTAs are incapable of colonizing a host, these resistant mutants are not expected to
survive in vivo. If they do not, then the null mutants are not a factor for resistance in animals.
As we have pointed out above, there are numerous other pathways that contain conditionally
essential enzymes linked to virulence-factor expression. Many of these enzymes could be good
antibiotic targets provided that the major mechanism for resistance involves deletion of the
pathway, and results in the production of avirulent organisms. The recent discovery of a small
molecule that inhibits a conditionally essential step in a virulence factor pathway provides a
starting point for investigating this novel antibacterial strategy.

Outlook
Extensive work over several decades has illuminated many of the roles of TAs in Gram-positive
bacteria and has firmly established their importance in bacterial physiology. A better
understanding of the WTA biosynthetic pathway has been aided by both biochemical and
genetic studies, and most of the steps in the B. subtilis and S. aureus WTA biosynthetic
pathways have been reconstituted in vitro by using synthetic substrates. A small molecule
antibiotic that targets WTA biosynthesis in S. aureus was recently discovered by utilizing the
recent genetic and biochemical advances in this field, and will make possible studies to evaluate
WTA biosynthesis as a pathway for therapeutic intervention. Positive outcomes from these
studies would validate this class of virulence factors as antibacterial targets and provide further
impetus for their study and exploitation.

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Acknowledgments
This work was supported by the NIH (1P01AI083214 and 5R01M078477 to S.W., and F3178727 to J.G.S.), a Mary
Fieser Postdoctoral Fellowship to J.C., and a training grant to T.C.M (T32-AI07061-30).

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Figure 1.
Simplified depiction of Gram-positive and Gram-negative bacterial cell envelopes. Gram-
negative organisms have a distinct periplasm; Gram-positive organisms do not, but recent
studies have suggested that they have a periplasmic-like compartment between the plasma
membrane and the base of the peptidoglycan layers.[3] Proteins are omitted from the depictions
for clarity. Membrane-embedded, membrane-anchored, and peptidoglycan-associated proteins
are abundant in the cell membranes of both Gram-positive and Gram-negative organisms. LTA:
lipoteichoic acid; LPS: lipopolysaccharide; WTA: wall teichoic acid.

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Figure 2.
Representative chemical structures of wall teichoic acids from different Gram-positive bacteria
(m = 1–3 and n = 20–40).

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Figure 3.
Genetic organization of wall teichoic acid biosynthetic genes; tag: teichoic acid glycerol; tar:
teichoic acid ribitol. Adapted from Qian et al.[48] (//: number of nucleic acids between genes
if > 120 base pairs).

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Figure 4.
Differences in wall teichoic acid biosynthesis for: A) B. subtilis 168, B) B. subtilis W23, and
C) S. aureus.

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Figure 5.
Proposed role for the distinct TarK and TarL WTA polymers. A) TarK and TarL make
electrophoretically distinct WTA polymers. PAGE analysis of WTAs extracted from various
S. aureus strains shows that poly(ribitol phosphate) polymer length corresponds to gene
regulation. The agr system directly or indirectly represses the expression of TarK, leading to
an increase in WTA polymer length. RN4220 has a partial defect in agr while RN450 has a
fully functional agr system (agr+). TarK makes a short secondary polymer, K-WTA, while
TarL makes a longer primary polymer, L-WTA. The L-WTA was extracted from a ∆tarK
mutant of S. aureus RN4220. K-WTA was extracted from an S. aureus RN4220 mutant over-
expressing tarK in a ∆tarL background.[61] B) Expression of the shorter K-WTA at low cell
density might allow for increased accessibility of adhesins, leading to a pro-adhesion state;
whereas, the expression of the longer L-WTA at high cell density (mediated by the activation
of agr) might lead to a low-adhesion phenotype.

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Figure 6.
Depiction of the primary Staphylococcus aureus wall teichoic acid (L-WTA) biosynthetic
pathway. Nonessential WTA pathway enzymes are colored green and their deletion leads to
an avirulent phenotype. Conditionally essential enzymes are colored red and their deletion is
lethal in a wild-type background but permitted in a ∆tarO or ∆tarA background. Following
intracellular assembly, the poly(ribitol phosphate) polymer is transported to the outside by a
two-component ABC transporter, TarGH, and then covalently linked through a phosphodiester
bond to the MurNAc sugars of peptidoglycan by an unidentified enzyme the biological and
genetic properties of which have not been established. In addition to TarI, J, and L, all S.
aureus strains contain a homologous set of enzymes (designated TarI′,J′ and K; Figure 3) that
directs the synthesis of a distinct WTA polymer (K-WTA); their cellular functions remain
incompletely understood.

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Figure 7.
Chemical structures of currently known inhibitors of wall teichoic acid synthesis and export
in Staphylococcus aureus. An inhibitor of DltA, which is involved in modification of both
WTAs and LTAs, has also been reported.[43]

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Figure 8.
General screening strategy for the identification of small-molecule inhibitors of conditionally
essential enzymes/targets in nonessential biosynthetic pathways; *: the mutant strain is
incapable of initiating polymer synthesis. In the described screen, the paired strain lacked the
first enzyme involved in WTA biosynthesis (TarO).

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