Download Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine

Novel Riboswitch Ligand Analogs as Selective Inhibitors
of Guanine-Related Metabolic Pathways
Jérôme Mulhbacher1, Eric Brouillette1, Marianne Allard1, Louis-Charles Fortier2, François Malouin1*,
Daniel A. Lafontaine1*
1 Département de biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada, 2 Département de microbiologie et d’infectiologie, Faculté de
médecine et sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada
Abstract
Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been
recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain.
Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical
for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 59-untranslated mRNA structure that
causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a
guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to
riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures.
Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity
against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only
achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial
strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a
different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the
administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the
possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and
selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as communityacquired bacterial infections have recently started to emerge.
Citation: Mulhbacher J, Brouillette E, Allard M, Fortier L-C, Malouin F, et al. (2010) Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related
Metabolic Pathways. PLoS Pathog 6(4): e1000865. doi:10.1371/journal.ppat.1000865
Editor: Michael R. Wessels, Children’s Hospital Boston, United States of America
Received October 8, 2009; Accepted March 22, 2010; Published April 22, 2010
Copyright: ß 2010 Mulhbacher et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Canadian Institutes of Health Research (CIHR) to DAL and the National Sciences and Engineering Research Council of
Canada (NSERC), Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, Canadian
Dairy Network, AAFC, PHAC, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research
Network to FM. A post-doctoral fellowship from NSERC and studentship from the Faculté des sciences of the Université de Sherbrooke were also provided to JM
and MA, respectively. DAL is a CIHR New Investigator scholar as well as a Chercheur-boursier Junior 2 from the Fonds de la recherche en Santé du Québec. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (FM); [email protected] (DAL)
027 [4,5,6]. This particular strain is spreading in North America
and Europe with currently little therapeutic solutions besides the
use of metronidazole and vancomycin, which are increasingly
associated with relapses and poor treatment outcome [7].
Previous attempts to discover alternative antibacterial drugs
targeting RNA were mainly based on a fortuitous interaction
between an exogenous ligand and its RNA target [1,8,9,10].
Metabolite-responsive riboswitches represent a novel solution to
MDR since they could be considered as antimicrobial targets
when agonistic ligands are employed as demonstrated for lysine,
thiamine pyrophosphate (TPP), flavin mononucleotide (FMN) and
guanine responsive riboswitches [1,11,12,13,14,15]. In the case of
lysine and TPP riboswitches, previously described ligand analogs
were reported to have a multitude of cellular effects in addition to
inhibition of gene expression via riboswitch binding [16,17,18,19].
Pleiotropic effects were also observed for compounds targeting the
guanine riboswitch and at least one analog was reported to be
possibly incorporated in DNA during replication [15,20]. Thus,
while it is of interest to select antibiotics that are chemically distinct
Introduction
Multiple drug resistance (MDR) has been a growing problem
during the last decade, partly due to excessive use of antibiotics in
human medicine and food animal production. MDR also stems
from the fact that drug design has been largely based on limited
chemical scaffolds leaving an opportunity for pathogens to
circumvent antibiotic action mechanisms [1]. Staphylococcus aureus
and Clostridium difficile are nosocomial pathogens responsible for a
significant mortality rate in hospitals and increased health care
costs [2]. Recently, community-acquired methicillin-resistant S.
aureus (MRSA) infections have emerged and are commonly
responsible for skin and soft-tissue infections that may rapidly
evolve in severe and life-threatening infections [3,4]. Moreover,
some emerging clones were shown to be resistant to vancomycin,
which is considered as the last chance antibiotic [5]. The pathogen
C. difficile has also dramatically increased the hospital-associated
deaths in recent years due to the MDR emergence and spreading
of the hypervirulent and high toxin-producing strain BI/NAP1/
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fact that purine riboswitches efficiently bind pyrimidines, it may be
possible to design novel antibiotics that bind to guanine
riboswitches and therefore inhibit bacterial growth.
Pyrimidine-based molecules that could fit into the guanine
riboswitch aptamer binding site were selected based on molecular
modeling of crystal structures [26,27] (Figure 2A). Using this
approach, we identified two pyrimidine compounds 2,5,6triaminopyrimidin-4-one (PC1) and 2,6-diaminopyrimidin-4-one
(PC2) that satisfied defined criteria such as geometrical constraint,
hydrogen bonding pattern and molecule planarity (Figure 2B). As
opposed to guanine, PC1 and PC2 lack one aromatic ring, making
them electronically distinct from guanine despite their similarity to
guanine in terms of H-bond donating and accepting potential.
Next, using the established in-line probing assay [25,28], we
monitored PC1/PC2-induced riboswitch conformational changes
(Figure 2C). In absence of ligand, several cleavage products that
map to previously reported single-stranded regions were observed
[25,28]. However, a cleavage reduction consistent with a
reorganization of the structure upon ligand binding was observed
in the core domain in presence of guanine (Figure 2C). In-line
probing assay with PC1 and PC2 instead of guanine showed an
identical cleavage pattern for both pyrimidine compounds and
guanine, suggesting that the core is reorganized similarly in
presence of these compounds, consistent with the recently reported
pyrimidine-bound riboswitch crystal structure [21].
To determine whether PC1 and PC2 repress gene expression,
we transformed B. subtilis with transcriptional fusions in which a
guanine riboswitch was fused to a lacZ reporter gene (Figure 2D).
When cells were grown in minimal medium with increasing
concentrations of PC1 and PC2, beta-galactosidase activity was
clearly repressed in a dose-dependent manner suggesting a
modulation of the guanine riboswitch gene regulation by both
molecules. We also performed growth inhibition experiments
using various concentrations of both PC1 and PC2 (Figure S2).
While growth inhibition was observed in minimal medium, no
such inhibition was observed using a richer medium such as
cation-adjusted Muller-Hinton broth (CAMHB). This selective
growth inhibition can be explained by PC1/PC2 inhibiting the
biosynthesis or transport of essential metabolites, which are
present in CAMHB but not in minimal medium. For instance, it
was recently shown that guanine-related compounds can only
inhibit B. subtilis growth in a minimal medium but not in Luria
broth; the growth inhibition was partly attributed to the
riboswitch-mediated repression of de novo purine synthesis [15].
Author Summary
During the last 30 years, bacterial resistance to antibiotics
has become a major problem. This situation is partly
because today’s antibiotics are mainly based on a limited
selection of chemical scaffolds, which makes it easier for
bacterial pathogens to quickly develop resistance against
new drug derivatives. This recurrent problem of multiple
drug resistance implies a constant need to search for novel
microbial targets and to modulate their activity using
artificial molecules. Riboswitches are newly discovered
gene regulatory elements that represent attractive targets
for antimicrobial drugs. Riboswitches are RNA structures
located in untranslated regions of messenger RNAs that
regulate the expression of genes involved in the transport
and metabolism of small metabolites. We have identified a
new antibiotic specifically targeting riboswitches found in
a subgroup of bacteria including Staphylococcus aureus
and Clostridium difficile, which are nosocomial pathogens
responsible for a significant mortality rate in hospitals, and
increased health care costs. The riboswitch controls the
expression of guaA that appears essential for virulence in
the mammalian host. A murine model was used as a proof
of principle to show that such an antibiotic could inhibit
the growth of S. aureus in a mammal. Our work provides
new insights into the discovery and design of novel
antimicrobial agents against bacterial pathogens.
from natural ligands to avoid cellular efflux or chemical
modification, it is important to consider that these chemical
differences will potentially help avoid patient toxicity due to offtarget binding. It is also important that the antibiotic provokes a
bacteriostatic or bactericidal effect either by targeting a single
gene, or a collection of genes, that is necessary for growth, or
essential for bacterial survival or virulence. Thus, because
modified pyrimidines can specifically bind the purine riboswitch
with affinities in the low nanomolar range [21], they make
excellent candidates to target purine riboswitches which are likely
potent drug targets given their role in regulating purine metabolic
pathways (Figure S1). For instance, the inactivation of the E. coli
GMP synthetase guaA leads to guanine auxotrophy [22] whereas
the inactivation of the B. subtilis IMP dehydrogenase guaB is lethal
[23]. Here we show that the guanine riboswitch in S. aureus and C.
difficile controls the expression of guaA and that this gene appears
essential for virulence in a murine model.
PC1-dependent bacterial growth inhibition requires guaA
to be riboswitch-regulated
Results
Pyrimidine-based antibiotics modulate the guanine
riboswitch activity
The S. aureus ATCC 29213 genome contains a unique guanine
riboswitch located immediately upstream of the xpt gene (Figure
S3). Very interestingly, RT-PCR experiments identified that the
riboswitch controls a four-gene operon consisting of xpt, pbuX, guaB
and guaA, thus placing guaA and guaB under the control of a
riboswitch in S. aureus (Figure S3). To determine if PC1 and PC2
have antibiotic activities by targeting the guanine riboswitch in S.
aureus, we performed antibiograms with PC1 and PC2 as well as
with three additional molecules having similar structures (compounds 3, 4 and 5). While compounds 3 and 5 are structurally very
close to PC1 and PC2, compound 4 is a guanine analog (Figure 3A).
Surprisingly, among the five compounds tested, only PC1 inhibited
bacterial growth in Muller-Hinton agar, which is consistent with its
ability to modulate riboswitch gene expression in B. subtilis
(Figure 2D). The absence of PC2 antibiotic activity is consistent
with the ,5-fold lower PC2-mediated gene expression modulation
in B. subtilis, which may result from the lower number of riboswitch-
Guanine-sensing riboswitches are members of the purine
riboswitch class, which also comprises adenine and 29-deoxyguanosine [24]. The guanine riboswitch negatively regulates transcription elongation at high guanine concentration in Bacillus
subtilis [25] (Figure 1A). The guanine aptamer is organized around
a three-way junction connecting three helices, in which a critically
important nucleotide is involved in a Watson-Crick base pair
interaction with the bound ligand [25] (Figure 1B). The ligand
binding site contains a cavity in which the metabolite is completely
surrounded by RNA contacts suggesting that most atomic
positions are important for the formation of the native ligandRNA complex [26,27]. By using appropriate aminopyrimidines, it
is also possible to recreate the correct network of hydrogen bonds
required to ensure proper complex formation as previously shown
for the adenine riboswitch [21]. Thus, by taking advantage of the
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Figure 1. The structure of the guanine riboswitch. (A) Scheme representing the guanine riboswitch secondary structures in absence (ON state)
or in presence (OFF state) of guanine. The formation of a guanine-riboswitch complex results in the adoption of an intrinsic terminator element that
prematurely stops transcription. (B) Consensus sequence and secondary structure of the guanine riboswitch aptamer. Nucleotides indicated in blue,
orange and green represent nucleotides that are conserved .90% and those colored in black are conserved .80% [28]. Nucleotides and lines in blue
and in green indicate interactions with the ligand via hydrogen bonding and base stacking, respectively. The cytosine 74 which confers ligand
binding specificity is shown in orange and the bound guanine is shown as a red rounded rectangle.
doi:10.1371/journal.ppat.1000865.g001
ligand interactions (Figure 2B). The binding affinity of PC1 suggests
that the guanine riboswitch can tolerate modifications on the ligand
pyrimidine ring that are not strongly deleterious for complex
formation (,100 nM vs ,5 nM for PC1 and guanine, respectively). The binding affinity of PC1 is very similar to that of
hypoxanthine, which is a naturally occurring guanine analog [25].
To explore the antibacterial activity spectrum of PC1, we used
several Gram-positive bacterial species which are potential human
pathogens containing guanine riboswitches. Of the 15 species
tested, 9 showed marked cellular growth inhibition, including
MDR strains and the C. difficile CD6 isolate representing the
hypervirulent NAP1/027 strain (Figure 3B). Interestingly, when
analyzing guanine riboswitch-regulated genes, we observed that all
PC1-responsive strains had guaA under riboswitch control whereas
the PC1-unresponsive ones did not employ riboswitch regulation
to control guaA. The best example of this correlation is that while
16S rDNA sequence analysis indicates that B. subtilis and Bacillus
halodurans are very closely related species [29], B. halodurans has a
guaA-controlled riboswitch and is sensitive to PC1 whereas B.
subtilis lacks a guaA-controlled riboswitch and is resistant to PC1.
Antibiogram results also showed that strains exhibiting pronounced MDR phenotypes are sensitive to PC1 suggesting that the
antimicrobial activity does not involve action mechanisms
common to other known antibiotics.
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Because our data suggest that PC1 acts by repressing the GMP
synthetase guaA, we reasoned that the PC1 inhibitory activity
should be reduced by GMP supplementation. S. aureus cells were
thus grown with or without supplemented GMP, and colony
forming units (CFU) were determined following serial microdilutions (Figure 3C). As predicted, bacterial growth inhibition was
relieved when GMP was provided to cells grown in presence of
PC1 for 2 h or 4 h, supporting the hypothesis that bacterial
growth inhibition is caused by the riboswitch-mediated guaA gene
repression that results in GMP cellular depletion.
The PC1 specificity was also confirmed using the Gramnegative bacterium Escherichia coli ATCC 35695, a strain that does
not contain guanine riboswitches. As expected, E. coli showed no
growth inhibition in presence of PC1 even when using strains
deficient for the AcrAB efflux system or having increased
membrane permeability (Figure S4). These results suggest that
the inability of PC1 to inhibit E. coli most probably results from
guaA not being under the control of a guanine riboswitch in E. coli.
Bactericidal activity and specificity of PC1
To further characterize the riboswitch inhibitory action
mechanism of PC1, S. aureus cells were grown in CAMHB in
presence of various ligand concentrations. We obtained a PC1
dose-dependent growth inhibition response characterized by a
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Figure 2. Guanine riboswitch agonists can be used to modulate gene expression. (A) Hydrogen bonds (left panel) and stacking interactions
(right panel) formed between the bound guanine and the guanine riboswitch [26,27]. Oxygen, nitrogen, and phosphorus atoms are in red, blue, and
yellow, respectively (left panel). Nucleotides follow the color scheme used in Figure 1B. Figures were prepared using PyMol (DeLano Scientific, San
Francisco, CA, USA). (B) Molecular recognition features for guanine (G) and predicted ones for PC1 and PC2. Blue and red arrows represent hydrogen
bond acceptors and donors, respectively. (C) In-line probing assays of the B. subtilis xpt riboswitch in the absence (2) or in the presence (+) of 1 mM
guanine (G), and 1 mM or 10 mM for both PC1 and PC2. Sites of substantial ligand-induced protections (positions 49–54) are assigned on the right by a
vertical bracket. Lanes NR, L and T1 correspond to molecules that were not reacted or that were partially digested by alkali or by RNase T1,
respectively. Guanines are identified on the left as molecular weight markers. (D) The beta-galactosidase activity of a xpt-lacZ transcriptional fusion
construct integrated in the genome of B. subtilis by recombination was assayed after 4 h of growth at 37uC in minimal medium in presence of the
indicated ligand concentrations. Each experiment was performed three times and the average as well as standard deviations are shown.
doi:10.1371/journal.ppat.1000865.g002
To analyze the PC1-mediated riboswitch inhibition on S. aureus
gene expression, we performed a transcriptomic microarray
analysis containing a selection of S. aureus genes involved in
different cellular processes such as virulence, secretion, general
stress responses, sensory/regulatory systems, antibiotic resistance,
iron transport and general biosynthesis [30] (Figure 4D). Among
the 468 genes analyzed, 72% were repressed by at least two folds
when S. aureus was treated with PC1 where the 16S rRNA gene
was the most repressed (Table S1). This result is consistent with a
riboswitch-mediated guaA gene expression inhibition leading to
GMP cellular depletion and RNA synthesis inhibition. This is
supported by the low expression of the guanine riboswitch operon
(xpt, pbuX, guaB and guaA) as well as the two DNA gyrase subunits
MIC of 0.625 mg/mL (Figure 4A). PC2 was also used and its
antibiotic activity was found to be less efficient than PC1, as
observed in B. subtilis (Figure S2). When compared to known
antibiotics, PC1 was found to have an extremely rapid bactericidal
activity similar to ciprofloxacin, one of the most bactericidal
antibiotics (Figure 4B). For instance, a 4 h treatment with PC1 led
to 6.6760.58 and 5.4261.02 log reductions in CFU/mL
compared to the untreated control for cultures of S. aureus ATCC
29213 and C. difficile CD6, respectively. When the same
experiment was repeated by adding either GMP or AMP to the
culture for 8 hours, bacterial growth was restored by a factor of
103 only in presence of GMP (Figure 4C), suggesting that PC1
growth inhibition activity is specific to guanine metabolism.
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Figure 3. S. aureus growth inhibition requires guaA to be under a riboswitch control. (A) Antibiograms performed on the S. aureus ATCC
29213 strain using 75 mg of PC1 (1), PC2 (2), 2-amino-4-hydroxy-6-methylpyrimidine (3), 9-methyl guanine (4), 5-bromo-6-methyl pyrimidine (5) and
PBS as control (6). Chemical structures of PC1 and PC2 are shown in Figure 2B. (B) PC1 antibiograms using bacterial strains with guanine riboswitches.
While PC1-insensitive strains do not have guaA under a riboswitch control (Bs: Bacillus subtilis, Ef: Enterococcus feacium, Lm: Listeria monocytogenes,
STRpy: Streptococcus pyogenes, STRd: Streptococcus dysgalactiae and STRu: Streptococcus uberis), PC1-sensitive strains control the guaA gene
expression via a riboswitch mechanism (Bh: Bacillus halodurans, STAa: S. aureus ATCC 29213, STAh: S. haemolyticus, SA228a: S. aureus resistant to betalactam, erythromycin, ciprofloxacin, gentamicin and tetracycline but susceptible to vancomycin, MRSA COL: methicilin resistant S. aureus COL, STAe:
S. epidermidis, Cb: Clostridium botulinum, CD630: C. difficile strain 630, CD6: C. difficile representing the hypervirulent NAP1/027 strain). (C) Influence of
GMP on PC1 bacterial growth inhibition. Spots from serial dilutions of S. aureus cultures in cation-adjusted Muller-Hinton broth (CAMHB) in absence
or presence of 600 mg/mL PC1 or 600 mg/mL PC1 supplemented with 100 mM GMP.
doi:10.1371/journal.ppat.1000865.g003
treated cells in a dose-dependent manner (data not shown) but its
low solubility prevents full recovery at higher doses. It is also
probable that GMP-related feedback inhibitory mechanisms were
responsible for some of the gene repression observed (as in the case
of the guanylate kinase gmk). Taken together, these results are
consistent with PC1 mainly acting through a riboswitch inhibition
mechanism that ultimately results in GMP cellular deprivation and
S. aureus growth inhibition.
(gyrA and gyrB), which were used as housekeeping gene controls
(Figure 4D). Of all the monitored genes in the microarray analysis,
only ahpF and ahpC, two genes involved in stress response
mechanisms, were activated by the PC1 treatment. However,
when S. aureus was treated with PC1 and GMP, the microarray
data showed an expression profile in which only 21% of the genes
surveyed were repressed. Whereas the housekeeping gyrase genes
were no longer repressed, the expression of the guanine riboswitch
operon was still reduced, consistent with PC1 binding the
riboswitch operon and inhibiting gene expression. The other
repressed genes mainly comprised those involved in virulence and
cell wall synthesis suggesting that the GMP supplemented cells
were still under stress [31], which is in agreement with the partial
growth rescue observed in Figure 4C. GMP is able to rescue PC1PLoS Pathogens | www.plospathogens.org
PC1 inhibits S. aureus growth in a murine model
Because our data showed that the growth repression activity of
PC1 is influenced by the presence of GMP, we decided to assess
the bactericidal activity of PC1 in a murine mastitis model of S.
aureus infection, which adequately represents the clinical context.
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Riboswitch Analogs Targeting Guanine Metabolism
Figure 4. PC1 shows bactericidal activity through cellular GMP depletion. (A) Minimal inhibitory concentrations (MIC) determination of PC1
and PC2 on S. aureus strain ATCC 29213 in CAMHB. MIC values of 600 mg/mL and 5000 mg/mL were obtained for PC1 and PC2, respectively. (B)
Bactericidal activity of PC1 and other known antibiotics against S. aureus as a function of time. For determination of the bactericidal effect of PC1,
bacteria were inoculated at 105 CFU/mL in absence (cont) or presence of 0.5 mg/mL ciprofloxacin (cipro), 0.5 mg/mL erythromycin (erythro), 1 mg/mL
vancomycin (vanco) and 600 mg/mL PC1. The concentration of each antibiotic corresponds to their MIC. (C) Bactericidal activity of PC1 against S.
aureus as a function of time in absence (cont) or presence of 600 mg/mL PC1, and in presence of PC1 with 100 mM GMP or AMP. (D) Relative
expression of S. aureus genes under the control of a guanine riboswitch when grown in presence of PC1 or PC1 with GMP. Results obtained in
presence of PC1 are normalized using xpt gene expression. Bacteria were inoculated at 108 CFU/mL in CAMHB in absence or presence of 600 mg/mL
PC1 with or without 100 mM GMP. Each experiment was performed three times and the average as well as standard deviations are shown.
doi:10.1371/journal.ppat.1000865.g004
Indeed, in addition to morbid nosocomial infections caused by S.
aureus, this bacterium is one of the major pathogen leading to
bovine mastitis, which is the most frequent and costly disease for
dairy producers with current antibiotic therapies usually failing to
eliminate infections from dairy herds [32]. The antimicrobial
activity of PC1 was therefore first tested on several S. aureus isolates
from mastitic cows, some of which having persisting chronic
infections (Figure 5A). A bactericidal effect of at least 4 orders of
magnitude was observed after a 4 h treatment with PC1. Next, to
ascertain that guaA was expressed in vivo and that this gene may be
important during infection, we monitored the expression level of
guaA by real-time PCR. When strain 1290 was grown either in
broth culture in vitro or when it was directly isolated from the
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mastitic milk of infected cows (M. Allard and F. Malouin, in
preparation), very similar expression levels were found for guaA and
the essential gene gyrB in both environments. This suggests that
PC1 could have an impact on guaA expression in vivo and thus be
used to treat S. aureus infections.
The proof of concept for the therapeutic efficacy of PC1 was
established in our murine model of S. aureus-induced mastitis [33].
At 4 h post-infection, different concentrations of PC1 were
administered to infected mice that were sacrificed 6 h later
(Figure 5B). When compared to mice that were not treated with
PC1, viable bacterial counts in the mammary gland were
drastically reduced in a dose-dependent manner. This strong
therapeutic effect was highly comparable to what we observed
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Figure 5. PC1 inhibits S. aureus clinical isolates in vitro and in vivo. (A) Fold reduction in viable counts (log10 CFU/mL) for the reference S.
aureus strain ATCC 29213 and for selected bovine isolates after a 4 h exposure to PC1 as compared to the untreated culture. Newbould 305 (ATCC
29740) and SHY97-3906 are bovine isolates from typical mastitis cases and isolates 3, 557 and 1290 were from cows with persisting intra-mammary
infections and chronic mastitis. (B) Bacterial counts (CFU) obtained from mice mammary glands 10 h post-infection with S. aureus. Mice mammary
glands were treated (intra-mammary administration) 4 h after infection with PBS with or without PC1 at 10, 50 or 100 mg/gland. Each dot represents
the CFU of each individual gland (n = 6–12) and the median value for each group is indicated by the bar. Statistical differences (P,0.05) between CFU
recovered from treated and untreated animals are shown by asterisks (non-parametric Kruskal-Wallis ANOVA with Dunn’s post test).
doi:10.1371/journal.ppat.1000865.g005
indicate that guaA is an important contributor to the survival of S.
aureus during infection and that it can be used as an antibiotic
target.
The major limitation to validate an antibiotic that targets
riboswitches is to evaluate the antibiotic specificity of action. In
this particular case, PC1 is not a broad-spectrum antibacterial
drug given that it does not target all bacteria containing guanine
riboswitches, but only those in which guaA is under the control of a
riboswitch. It is not excluded that other riboswitch-controlled
genes may participate in the PC1-dependent bacterial growth
inhibition (e.g., guaB), or that PC1 may bind other cellular targets,
which alone or in combination with the riboswitch-controlled gene
repression, would repress bacterial growth. Nevertheless, the
restricted nature of growth inhibition likely indicates that PC1
inhibits bacterial growth through riboswitch binding and not via
an alternative mechanism such as DNA incorporation (Figure 6).
For instance, when performing antibiograms using the guanine
analog 6-thioguanine, a general growth inhibition was observed in
E. coli and S. aureus (Figure S7), consistent with its incorporation
into DNA that perturbs the epigenetic pathway of gene regulation
[40]. The selective antibacterial activity of PC1 toward S. aureus
was also supported by the lack of apparent toxicity for mice treated
with the experimental compound at concentrations as high as
100 mg/gland with no sign of discomfort including vocalizations,
curved back, piloerection and hypothermia. There was also no
apparent cytotoxicity upon histological observations of mammary
tissues in PC1-treated mice compared to PBS-treated glands
(Figure S8).
Recently, the Breaker group used purine derivatives modified in
position 2 or 4 to target guanine riboswitches and found three
molecules that inhibited bacterial growth [15]. Among these
molecules, only one was able to repress the expression of a guanine
riboswitch-controlled reporter gene suggesting that at least two
molecules inhibited cell growth through a different mechanism of
action. It is possible that the mode of action of these two molecules
involves nucleic acids incorporation following ribosylation at
with known antibiotics. For example, amoxicillin decreased the
bacterial load in the mammary gland to a log10 median value of
3.97 CFU/g of gland at a dose of 50 mg/gland. Noteworthy, a
dose of 50 mg of amoxicillin would represent 100xMIC/g of gland,
whereas a similar dose would only represent a twelfth of the MIC/
g of gland for PC1. This result is consistent with the idea that PC1
is most efficient in the mammary gland environment suggesting
that the microaerobic condition (i.e., low oxidative environment)
of the mastitic milk[34] helps PC1 therapeutic efficacy. Consistent
with these results, we found that the potency of PC1 was
significantly increased by preventing its oxidation using a reductive
agent such as DTT in susceptibility tests in vitro (Figure S5).
Discussion
Despite previous large scale screen data suggesting that guaA is
not essential for S. aureus growth [23,35] in relatively rich media,
we show here that blocking guaA expression can lead to
bactericidal activity in various bacterial species. In support of
guaA for cell viability, it has been recently reported that mutations
occurring in guaA prevent Streptococcus suis [36] and Salmonella
thyphimurium [37] from properly infecting porcine and murine
models, respectively, suggesting that GMP bioavailability may be
reduced during host infection and that guaA is likely to be crucial
for bacterial infection in mammals. Thus, together with studies
showing the importance of guaA for bacterial growth in urine or
blood [38,39], our data suggest that mammalian infection sites
may significantly differ in their nutrient compositions from those
used in large scale screens [23,35], and that care should be taken
when assessing the ‘‘essentiality’’ of a gene. Furthermore, when
assessing whether S. aureus could develop resistance to PC1, no
resistant bacteria were obtained after more than 30 passages
suggesting that maintaining a functional guaA-regulated riboswitch
is a vital process (Figure S6). Taken together, the demonstration
that guaA expression is normally maintained in S. aureus grown in
vivo and the strong therapeutic effect resulting from PC1 treatment
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Riboswitch Analogs Targeting Guanine Metabolism
Figure 6. Scheme representing the action mechanism of PC1 on S. aureus guanine pathway. Genes highlighted in blue correspond to
those of the operon xpt-pbuX-guaB-guaA that is controlled by the guanine riboswitch. Red lines indicate genes that are inhibited by PC1 via its
binding to the guanine riboswitch. Of these four inhibited genes, guaA and guaB are known to be critical for guanine nucleotide biosynthesis
[22,23,36,37,38,39] and their inhibition very likely lead to the repression of GMP synthesis and most probably of RNA and DNA production.
doi:10.1371/journal.ppat.1000865.g006
Canadian Council on Animal Care were respected during all the
procedures.
position 9 of the purine analog, as demonstrated for 6-thioguanine.
It is also interesting to mention that the antibiotic activity was only
observed using B. subtilis strains cultivated in minimal medium
whereas no growth inhibition was detected in rich media. In our
study, we selected guanine riboswitch ligands that cannot be
ribosylated to prevent alternative modes of action, and favored a
pyrimidine compound (PC1) that retained most of the key
functional groups. By testing various bacterial species, we observed
that the bactericidal activity of PC1 was only seen against bacteria
using a guanine riboswitch to control guaA expression, and this
even when bacteria were grown in a rich medium. This
demonstrates that analog binding to riboswitch aptamers is not
the only determinant to achieve selective and efficient bactericidal
effects.
This study shows for the first time that antibiotics targeting
riboswitches may be efficient to kill bacterial pathogens in vitro as
well as in mammalian infection models. We found here that the
selective bacterial killing of PC1 is only achieved when guaA, a
GMP synthetase, is under the control of the riboswitch and when
the antimicrobial agent cannot be ribosylated. The narrow
spectrum of activity we demonstrated here for PC1 is very
interesting since two of the target bacteria, S. aureus and C. difficile,
are among the most problematic nosocomial pathogens. The
spread of MDR in those bacterial species also stresses the
importance to develop new antibiotics that avoid current
mechanisms of resistance. The use of narrow spectrum drugs
should be encouraged whenever possible to reduce any selective
pressure for resistance in non-targeted bacteria. Here, we also
showed that the development of resistance toward PC1 is likely to
be infrequent.
Reagents
4-hydroxy-2,5,6-triaminopyrimidine (PC1), 2,4-diamino-6-hydroxypirimidine (PC2) and 9-methylguanine were purchased from
Fluka. 2-amino-5-bromo-6-methyl-4-pyrimidol and 2-amino-4hydroxy-6-methylpyrimidine were purchased from Aldrich.
Strategy of ligand selection
Our ligand selection took into account guanine binding
requirements [25] and crystal structure interactions [21,26,27].
Planar molecules were selected to preserve stacking interactions
with adenines 21 and 52 in the guanine aptamer binding site. One
of our important selection criteria was to avoid the presence of
functional groups that could serve as a ribosylation site in purine
or pyrimidine analogs that would allow subsequent nucleic acid
incorporation and non-specific antibiotic effect. Successfully
identified molecules were drawn using Chem3D Pro (CambridgeSoft) and docked onto the guanine aptamer crystal structure (PDB
1U8D). The in silico procedure was important to validate aptamerligand interactions and to avoid sterical obstructions that would
perturb ligand binding.
Transcription of RNA
For the production of guanine riboswitch aptamers, DNA
templates were prepared from partial duplexes and transcribed
using T7 RNA polymerase as previously described [28]. The
aptamer sequences used in this study are based on the genomic
sequence to which a GCG sequence is added to the 59 side to allow
high transcription yield and to minimize the 59 heterogeneity [28].
Methods
Ethics statement
In-line probing assays
The institutional ethics committee on animal experimentation
of the Faculté des sciences of the Université de Sherbrooke (QC,
Canada) approved these experiments and the guidelines of the
[59-32P] RNA molecules were incubated for 96 h at 25uC in
50 mM Tris-HCl buffer, pH 8.5, 20 mM MgCl2 and 100 mM
KCl in absence or in presence of indicated ligand concentrations.
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Riboswitch Analogs Targeting Guanine Metabolism
4 h after infection with PBS or PBS with 10, 50 and 100 mg/gland
of PC1 and mice were sacrified 6 h later for mammary gland
sampling and homogenization. The tissues used for CFU counts
were homogenized in 2 mL of PBS and the bacterial content was
evaluated by serial logarithmic dilutions on agar. The detection
limit was 100 CFU/g of gland.
The reactions were stopped with a 97% formamide solution
containing 10 mM EDTA and samples were purified by
electrophoresis in 10% polyacrylamide gels (acrylamide:bisacrylamide; 19:1) containing 8 M urea. Gel were dried and exposed to
Phosphor Imager screens.
beta-galactosidase assays
Supporting Information
Regulation of the beta-galactosidase reporter gene expression in
presence of PC1 or PC2 was determined using an xpt-lacZ
transcriptional fusion construct integrated in the genome of B.
subtilis by recombination. The beta-galactosidase activity was
measured after 4 h of growth at 37uC in minimal medium in
absence or presence of the indicated ligand concentrations [41].
Figure S1 Guanine and guanine-related metabolic pathways in
different bacterial species. Guanine riboswitch-regulated genes are
shown using a color scheme where Bacillus subtilis, Clostridium difficile
and Staphylococcus aureus are indicated in green, orange and blue,
respectively. Dashed lines represent metabolite membrane transporters. The synthesis of GMP is highly dependent upon guaA and
guaB, which respectively encodes a GMP synthetase and an IMP
dehydrogenase. While guaB has been shown to be essential in B.
subtilis [23], mutations in guaA have been reported to make cells
auxotroph for guanine [22]. The importance of guaA and guaB has
also been observed during bacterial infections in murine and
porcine models [36,37]. Thus, guaA and guaB encode two enzymes
critical for guanine nucleotide biosynthesis and reduction of their
expression via riboswitch action is very likely to produce bacterial
growth inhibition.
Found at: doi:10.1371/journal.ppat.1000865.s001 (0.32 MB TIF)
Antibiogram assays
Bacteria were inoculated at 105 CFU/mL in melted MullerHinton agar. After agar medium was solidified, six wells of 4 mm
in diameter were made and filled with 10 mL of the tested
molecules (5 mg/mL). Plates were incubated for 16 h at 37uC.
Antibiotic minimal inhibitory concentrations
The minimal inhibitory concentration (MIC) of PC1 and PC2
against S. aureus strain ATCC 29213 was determined using a
microdilution method in 96-well plates [30]. Bacteria were
inoculated at 105 CFU/mL and incubated at 37uC for 24 h in
cation-adjusted Muller-Hinton broth (CAMHB). Bacterial growth
was detected by measuring the OD at 595 nm on a microplate
reader.
Figure S2 B. subtilis growth is inhibited by PC1 acting as a
guanine riboswitch antibiotic. (A) Minimal inhibitory concentrations (MIC) of PC1 and PC2 against B. subtilis. The MICs were
determined using a microdilution method in 96-well plates.
Bacteria were inoculated at 105 CFU/mL in cation-adjusted
Muller-Hinton broth (CAMHB) or in minimal media (MM) and
incubated at 37uC for 24 h. MIC values of 1250 mg/mL and
5000 mg/mL were respectively obtained for PC1 and PC2 in MM,
but no growth inhibition was observed in CAMHB. (B) PC1
modulates riboswitch activity in CAMHB in a dose-dependent
manner. Beta-galactosidase activity of a xpt riboswitch-lacZ
transcriptional fusion construct integrated by recombination in
the genome of B. subtilis was assayed after 4 h of growth at 37uC in
CAMHB in the absence or presence of the indicated ligand
concentrations. This confirms that PC1 can modulate riboswitch
activity in a relatively rich media such as CAMHB and not only in
a minimal media (Figure 2D), where growth inhibition is observed.
Each experiment was performed three times and the average as
well as the SD are shown.
Found at: doi:10.1371/journal.ppat.1000865.s002 (0.39 MB TIF)
Antibiotic bactericidal activity
Time-kill experiments were performed for the determination of
the bactericidal effect of test antibiotics. Bacteria were inoculated
at 105 CFU/mL in CAMHB in absence or presence of the
antibiotic at its MIC with or without 100 mM GMP. Bacterial
permeability to GMP was increased by adding 0.002% Triton X100. At several time points, bacteria were sampled and serially
diluted before spreading on tryptic soya agar (TSA) plates for CFU
determinations. Plates were incubated for 24 h at 37uC.
Transcriptomic microarray
Bacteria were inoculated at 108 CFU/mL in CAMHB in
absence or presence of 600 mg/mL PC1 or 600 mg/mL PC1
supplemented with 100 mM GMP. After 30 min of growth, RNA
was extracted and 2.5 mg of RNA were submitted to reverse
transcription to generate fluorescent probes through an aminoallyl
cDNA labeling procedure before being hybridized on the
microarray [30].
Figure S3 Schematic representation of the S. aureus xpt-pbuXguaB-guaA operon controlled by the guanine riboswitch. (A) The
predicted number of nucleotides for each intergenic region is
shown. (B) RT-PCR of intergenic regions performed using total
RNA. Note that an amplification product is obtained in all cases
indicating that xpt, pbuX, guaB and guaA are included in an operon
controlled by the guanine riboswitch. RNA was extracted from
lysate using a Quiagen RNeasy kit and treated with DNase I in
presence of RNase inhibitors. Following this, 1 mg was used for
reverse transcription with 200 units of SuperScript II (Invitrogen),
using 100 pmoles of a DNA oligonucleotide used as a primer. The
reaction was performed at 42uC for 1 h and used in a PCR
reaction using an appropriate forward primer. Lane L represents a
100 bp ladder with the number of base pairs indicated for each
band. Lanes xpt-pbuX, pbuX-guaB and guaB-guaA represent PCR
reactions amplifying corresponding intergenic regions.
Found at: doi:10.1371/journal.ppat.1000865.s003 (1.02 MB
TIF)
Murine mastitis model
Experimental conditions used here for the mastitis model were
previously optimized for S. aureus Newbould and antibiotic
treatment [42]. CD-1 lactating mice (Charles River, St. Constant,
Canada) were used 12 to 14 days after offspring birth and typically
weighed 35 to 40 g. Pups were removed 1 h before bacterial
inoculation of mammary glands and a mixture of ketamine/
xylazine at 87 and 13 mg/kg of weight, respectively, was used for
anesthesia of lactating mice. A 100 ml syringe with a 33-gauge
blunt needle was used to inoculate both L4 (on the left) and R4 (on
the right) abdominal mammary glands. These large glands
constitute the fourth pair found from head to tail. Each udder
canal was exposed by a small cut at the near end of the teat under
a binocular and 100 mL of bacterial suspension (1 CFU/mL) was
injected through the orifice. Mice mammary glands were treated
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Riboswitch Analogs Targeting Guanine Metabolism
Figure S4 PC1 does not inhibit the growth of the Gram negative
bacterium E. coli, which does not naturally contain guanine
riboswitches. The MIC of PC1 and other known antibiotics
against E. coli ATCC 35695 were determined using a broth
microdilution method. As expected, PC1 does not show any
antibiotic activity toward E. coli ATCC 35695, most probably
because of the absence of a guanine riboswitch. However, to
exclude the possibility that the lack of inhibitory activity is due to a
poor cell penetration of PC1 into E. coli or to an active efflux of this
compound out of the cells, we also tested PC1 activity against two
isogenic E. coli mutants. While E. coli ATCC 35695 is a standard
strain, AcrAB is deficient for the multidrug efflux pump AcrAB
[43] and Imp has an increased membrane permeability [44]. Our
results show that there is still no inhibitory activity of PC1 against
any of the mutants. Please note that the Imp increased membrane
permeability is confirmed by the antibiotic activity of vancomycin,
which is a large glycopeptide molecule for which Gram negative
bacteria are normally impermeable. Besides, the reduced efflux
activity of AcrAB is verified from the ability of erythromycin to
inhibit bacterial growth. Erythromycin is also able to inhibit Imp
bacterial growth due to the increased membrane permeability.
These results indicate that the lack of PC1 antibiotic activity
toward E. coli is not due to its active efflux by the bacteria or its
inability to pass through the cell membrane. All concentrations are
in mg/mL and the chemical structure for PC1 is shown in
Figure 2B.
Found at: doi:10.1371/journal.ppat.1000865.s004 (0.12 MB TIF)
antibiotics, ciprofloxacin and rifampicin, were added to the
histogram. High level resistance to ciprofloxacin and rifampicin
is rapidly selected (within 5 daily passages) in S. aureus. Such rapid
development of resistance for the traditional drugs is consistent
with the selection of known single point mutations each able to
provide a decrease in drug affinity for the bacterial cell target.
There are at least 2 known point mutations in GyrA conferring
resistance to ciprofloxacin in addition to possible over-expression
of the NorA efflux pump system also occurring through mutations
(at least 3 possible mutations) [46] and at least 17 possible different
mutations in RpoB enabling resistance to rifampicin have been
documented [47]. The absence of resistance observed in presence
of PC1 is probably because reestablishing guaA gene expression in
the presence of PC1 requires multiple mutational steps thus
reducing the frequency of resistance development and/or that
maintaining a functional riboswitch is a vital process that does not
allow bacteria to bypass PC1 antibiotic action. PC1 experiments
were performed three times and the average as well as the SD are
shown.
Found at: doi:10.1371/journal.ppat.1000865.s006 (0.46 MB TIF)
Figure S7 Antibiograms performed on strains of E. coli ATCC
35695 and Methicilin Resistant S. aureus strain COL. E. coli ATCC
35695 (A) and S. aureus strain COL (B) were grown in absence (well
#1) or in presence of 7.5 mg (well #2) or 15 mg 6-thioguanine (well
#3). Please note that 6-thioguanine is able to be ribosylated (C)
and incorporated in DNA[40], which probably explains its
riboswitch-independent antibiotic activity toward both E. coli
and S. aureus.
Found at: doi:10.1371/journal.ppat.1000865.s007 (3.01 MB TIF)
DTT increases the antibiotic activity of PC1 probably
by preventing its oxidative self-condensation. (A) MICs were
determined using a microdilution method in 96-well microplates in
absence or presence of DTT at the concentrations shown. Bacteria
were inoculated at 105 CFU/mL and incubated at 37uC for 24 h
in CAMHB. Please note that the MIC is decreased by a factor of
,40x in the presence of 0.05% DTT. At low PC1 concentrations,
it can be observed that DTT does not inhibit bacterial growth by
itself. (B) Schematic representation of the probable oxidative selfcondensation of PC1 that could be prevented by DTT. Previous
studies have shown that 4,5-diaminopyrimidines can produce
insoluble and deeply-colored orange substances such as pyrimido[5,4-g]- and pyrimido[4,5-g]pteridines by oxidative selfcondensation [45]. Given that PC1 is structurally very similar to
4,5-diaminopyrimidines and that it produces an orange precipitate
over time, it is likely that PC1 also self-condenses by air oxidation.
This is consistent with the observation that a reductive agent such
as DTT can slow down the formation of the precipitate (data not
shown).
Found at: doi:10.1371/journal.ppat.1000865.s005 (0.37 MB TIF)
Figure S5
Figure S8 Histology of mice mammary glands treated with PC1.
Mice were either injected with (A) 100 mL PBS or with (B) 100 mL
PBS containing 100 mg of PC1. The treatment was allowed for 6 h
and mammary glands were excised, fixed in 4% formaldehyde for
24 h at room temperature and embedded in paraffin wax.
Hematoxylin-eosin staining was done on sections of 5 mm
thickness. Magnifications on the pictures are 2006. The histology
study reveals that there is no observable damage to the gland
following PC1 injection.
Found at: doi:10.1371/journal.ppat.1000865.s008 (5.90 MB TIF)
Table S1 Transcriptomic microarray showing the relative
expression of S. aureus genes as a function of PC1 and GMP.
Found at: doi:10.1371/journal.ppat.1000865.s009 (0.08 MB XLS)
Acknowledgments
We thank members of the Lafontaine laboratory and Eric Marsault for
helpful discussions and Dr. Alain Lavigueur for critical reading of the
manuscript and Gilles Grondin for the histological studies.
Figure S6 Serial passages in the presence of sub-inhibitory
concentrations of test antibiotics demonstrating the inability of S.
aureus to develop resistance toward PC1. The MIC of test
compounds against S. aureus strain ATCC 29213 recovered from
broth cultures containing sub-inhibitory concentrations of antibiotics (0.25X of MIC) was determined every 5 passages for up to 30
passages. As a comparison, results obtained with two known
Author Contributions
Conceived and designed the experiments: JM LCF FM DAL. Performed
the experiments: JM EB MA FM. Analyzed the data: JM LCF DAL.
Contributed reagents/materials/analysis tools: LCF FM DAL. Wrote the
paper: JM LCF FM DAL.
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SUPPLEMENTARY TABLE 1
Experimental conditions (Test vs control Cy3 vs Cy5, PC1 600!g/mL alone or with 100 !M GMP, incubation time 30min, S aureus ATCC29213 inoculation 10e8 CFU/mL)
ID SACOL Gene
General Function
Gene Description
1254
1159
1160
672
1158
272
18
144
149
2025
2026
2218
1371
2242
2213
1449
145
1734
2340
2688
140
1908
150
2694
936
1490
142
1396
20
938
2363
2271
1194
1026
1373
1195
1079
2347
2270
457
141
1448
1385
1173
276
22
950
2718
935
2272
700
195
1963
679
306
148
494
689
1742
2023
1320
1196
766
860
762
151
701
1812
5
136
2287
2577
799
690
96
1066
2054
992
2362
163
765
2413
154
277
699
2712
95
1637
743
703
2394
1272
2067
2293
86
758
541
127
Control
Energy metabolism
Energy metabolism
Virulence regulator
Energy metabolism
Stress response
Nucleic acid metabolism
Virulence capsule
Virulence capsule
Virulence regulator
Virulence regulator
Nucleic acid metabolism
Nucleic acid metabolism
Nucleic acid metabolism
Nucleic acid metabolism
Energy metabolism
Virulence capsule
Energy metabolism
Membrane transport
Virulence biofilm
Virulence capsule
Energy metabolism
Virulence capsule
Virulence
Cell wall/Cell division
Cell wall/Cell division
Virulence capsule
Cell wall/Cell division
Regulation
Cell wall/Cell division
Energy metabolism
Membrane transport
Cell wall/Cell division
Stress response
Stress response
Cell wall/Cell division
Nucleic acid metabolism
Membrane transport
Membrane transport
Stress response
Virulence capsule
Energy metabolism
Energy metabolism
Virulence enzyme/toxin
Virulence enzyme/toxin
Regulation
Membrane transport
Membrane transport
Cell wall/Cell division
Membrane transport
Membrane transport
Membrane transport
Membrane transport
Membrane transport
Membrane transport
Virulence capsule
Energy metabolism
Membrane transport
Energy metabolism
Virulence regulator
Energy metabolism
Cell wall/Cell division
Virulence regulator
Virulence enzyme/toxin
Virulence enzyme/toxin
Virulence capsule
Nucleic acid metabolism
Virulence regulator
Nucleic acid metabolism
Virulence capsule
Virulence regulator
Stress response
Membrane transport
Membrane transport
Virulence regulator
Cell wall/Cell division
Stress response
Membrane transport
Energy metabolism
Membrane transport
Virulence regulator
Membrane transport
Energy metabolism
Stress response
Cell wall/Cell division
Stress response
Virulence adhesin
Stress response
Cell wall/Cell division
Nucleic acid metabolism
Energy metabolism
Membrane transport
Membrane transport
Stress response
Membrane transport
Energy metabolism
Virulence regulator
Membrane transport
Ribosomal RNA 16S
Succinate dehydrogenase
Succinate dehydrogenase
Staphylococcal accessory regulator A
Succinate dehydrogenase
Hypothetical protein
Adenylsuccinate synthetase
Capsular polysaccharide biosynthesis
Capsular polysaccharide biosynthesis
Accessory gene regulator protein C
Accessory gene regulator protein A
Adenylate kinase
GMP reductase
Xanthine/uracil permease
DNA-directed RNA polymerase, alpha subunit
2-oxoglutarate dehydrogenase
Capsular polysaccharide biosynthesis
Glyceraldehyde 3P deshydrogenase
Sodium/glutamate symporter
Intercellular adhesion regulator
Capsular polysaccharide biosynthesis
Fumarate hydratase
Capsular polysaccharide biosynthesis
Triacylglycerol lipase
DltB protein, lipoteichoic acid synthesis
Penicillin-binding protein 2
Capsular polysaccharide biosynthesis, UDP-N-acetylglucosamine 2-epimerase
Membrane protein, putative (methicillin/oxacillin resistance-related protein)
Sensory box histidine kinase, two component system
DltD protein, lipoteichoic acid synthesis
L-lactate permease
Molybdenum ABC transporter, permease protein
Penicillin-binding protein 1
Conserved hypothetical protein (toxic anion resistance)
Conserved protein
Phospho-N-acetylmuramoyl-pentapeptide-transferase
Amidophosphoribosyltransferase
Drug resistance transporter, EmrB/QacA
Molybdenum ABC transporter, ATP-binding protein
Conserved protein
Capsular polysaccharide biosynthesis
2-oxoglutarate dehydrogenase
Aconitate hydratase
Alpha-hemolysin
Diarrhoeal toxin
Unknown function, two component system
Na/H antiporter
Anion transporter family protein
D-alanine-activating enzyme / D-alanine-D-alanyl carrier protein ligase
Molybdenum ABC transporter, molybdenum-binding protein
ABC transporter
Maltose ABC transporter, permease protein
Proline permease
Na+/H+ antiporter component, MnhA component putative
ABC transporter, ATP-binding protein
Capsular polysaccharide biosynthesis, galactosyltransferase
NADH dehydrogenase I, F subunit
Manganese ABC transporter, ATP-binding protein
Citrate synthase
Accessory gene regulator protein B
Glycerol kinase
UDP-N-acetylmuramoylalanine-D-glutamate ligase
Two-component response regulator systems involved in virulence
Thermonuclease
Probable hemolysin
Capsular polysaccharide biosynthesis, UDP-N-acetylglucosamine 2-epimerase
Nucleoside permease NupC putative
Regulator of toxin, Rot
DNA gyrase, B subunit
Capsular polysaccharide biosynthesis
Staphylococcal accessory regulator R
Dehydrosqualene synthase
Possible ferrichrome ABC transporter, lipoprotein
Manganese ABC transporter, ATP-binding protein
Staphylococcal accessory regulator S involved in virulence
Autolysis and methicillin resistant-related protein
RNA polymerase sigma factor B
Oligopeptide ABC transporter, permease protein
Malate:quinone oxidoreductase
Drug transporter, putative
Two-component response regulator systems involved in virulence
Drug resistance transporter, EmrB/QacA subfamily
Aldehyde dehydrogenase
Hypothetical protein
Penicillin-binding protein 4
drp35 protein, induced by detergents that perturb membrane integrity
Immunoglobulin G binding protein A
Chaperone protein
bacitracin resistance protein
Conserved hypothetical protein
Nitrate reductase
Dipeptide transport operon repressor
Potassium-transporting ATPase, B subunit
NAD/NADP octopine/nopaline dehydrogenase, Csb22
Drug transporter, putative
1-phosphofructokinase, PTS system
StageV sporulation protein G homolog
Phosphonate ABC transporter, ATP-binding protein
ARNr16S
sdhA
sdhB
sarA
sdhC
purA
cap5I
cap5N
agrC
agrA
adk
guaC
rpoA
sucA
cap5J
gapA2
gltS
icaR
cap5E
fumC
cap5O
lip
dltB
pbp2
cap5G
fmtC
yycG
dltD
pbpA
mraY
purF
Csb12
cap5F
sucB
hla
yukA
yycl
mnhF
dltA
putP
cap5M
nuoF
mntB
gltA
agrB
glpK
murD
saeR
nuc
cap5P
rot
gyrB
cap5A
sarR
crtM
sstD
mntA
sarS
fmt
sigB
oppC
mqo
saeS
pbp4
drp35
spa
dnaK
bacA
narH
codY
kdpB
Csb22
fruK
spoVG
PC1 conditions
PC1+GMP
Expression mean
Expression mean
(log2 test vs control) (log2 test vs control)
-2,276315179
0,124166548
-2,266763775
0,209533678
-2,205044554
0,100398981
-2,139859562
-1,153239324
-1,971835092
0,249345701
-1,907698397
-1,606319039
-1,846259849
0,04037557
-1,815654842
-2,889059757
-1,784005645
-2,821112981
-1,777667869
-1,382458034
-1,774362501
-1,490145879
-1,76442508
0,589716533
-1,760380429
-2,238302653
-1,751125689
-2,552369633
-1,741631903
0,447415688
-1,71330638
-0,192127347
-1,688078618
-2,441351897
-1,660960927
-0,140416383
-1,654312419
0,171927784
-1,645879568
-1,451540589
-1,641499835
-3,194995211
-1,623326637
-0,820555424
-1,609147137
-2,661417701
-1,578259669
-1,2430572
-1,570719399
-0,449325579
-1,552597598
0,220384095
-1,547451826
-2,918010858
-1,543381403
-1,35858805
-1,521387703
-0,862262076
-1,51945954
-0,338891055
-1,503772748
3,021915309
-1,503506466
-1,023331258
-1,491845527
-0,378792682
-1,487932833
-0,020179165
-1,487592464
-1,758507247
-1,477075831
-0,325974743
-1,471858151
-2,874759702
-1,471456632
-0,080242866
-1,46874895
-1,013976036
-1,462406194
0,632822
-1,460616418
-3,008400868
-1,456317648
-0,443859366
-1,45481507
0,237683636
-1,444455408
-0,918134001
-1,405294647
-1,140579126
-1,404933905
-0,857612223
-1,394796477
-0,43906089
-1,387438492
-0,962162731
-1,386853591
-0,815893941
-1,383249994
-0,93269218
-1,378179532
-1,354195229
-1,369364431
-0,401189163
-1,36871877
-0,595243894
-1,365938987
-4,194934517
-1,346763512
-0,378267351
-1,344548654
-1,98088575
-1,332516284
-0,221305303
-1,327342135
-6,616648898
-1,322817966
0,082200023
-1,301299694
-1,169153088
-1,298185926
0,326744321
-1,292737977
-0,40174189
-1,29208997
-0,684152776
-1,290659635
-1,861763669
-1,284205984
-1,247008645
-1,27167011
-2,605659184
-1,268461061
-1,416809677
-1,266421525
-0,6142797
-1,266320558
-0,239525421
-1,265093242
-3,141989055
-1,264155803
-1,029776045
-1,260161111
-0,348047043
-1,256011572
0,444778626
-1,251266351
-6,496446924
-1,244756232
-0,697355349
-1,240243621
-0,750287521
-1,240003729
-0,407054575
-1,239779053
-1,282216578
-1,222197975
0,016302743
-1,221760292
-0,899877807
-1,20563678
-0,440077857
-1,197896449
-1,190868881
-1,19078249
-0,06355122
-1,189078032
-1,002982146
-1,18858172
-1,0959026
-1,187461263
0,021739335
-1,183902588
-2,689550181
-1,181735612
0,130788454
-1,18064911
-0,441889572
-1,18004176
-1,239969327
-1,180035641
-0,324282691
-1,17453011
0,147534267
-1,172527125
-2,091974307
-1,170002475
-1,246505442
-1,163177463
-0,527297627
-1,162133852
0,233116892
-1,161574661
-0,299397213
-1,156375005
-0,463504769
1197
2266
2125
1609
1639
1114
1970
754
2291
2352
809
2582
2430
1427
2022
1664
1193
1328
1657
1103
2576
1790
2055
1638
2068
2392
1994
1198
1912
608
1105
1262
968
1898
1414
78
1680
1142
2708
1184
1535
2691
2572
620
209
121
757
29
2006
459
2656
1534
1199
2151
248
969
2134
1613
1424
1825
2066
660
1715
695
2581
2004
1357
2016
2505
1055
162
2566
1741
1969
1021
2712
810
544
1944
1777
2676
2342
98
2325
245
2399
1085
2422
1398
1421
246
1468
1743
1716
1054
2659
2421
2564
416
2057
688
1057
609
712
2431
divlB
moeA
pbp3
hrcA
sspB2
norA
tcaA
hld
ftsL
glnR
pdhB
crtN
murC
rsbW
grpE
kdpA
narl
ftsA
Csb33
sdrC
pdhD
sucC
spsA
cbf1
plc
csbD/Csb8
isdD
ext
srrA
icaB
copA
proP
coa
deoD
pbuX
arcB
srrB
ftsZ
femD
lrgB
spsB
kdpC
adh
hemB
tagG
groEL
sasG
sspC
mmpL
icd
purB
drp35
prsA
htrA
sasA/sdrD
corA
sirB
lyt
nirR
hlgB
lytR
hemD
menB
aur
hlgC
feoB
rsbU
mntC
sspA
sdrD
Cell wall/Cell division
Membrane transport
Stress response
Cell wall/Cell division
Stress response
Membrane transport
Virulence
Membrane transport
Virulence
Cell wall/Cell division
Regulation
Cell wall/Cell division
Membrane transport
Membrane transport
Virulence enzyme/toxin
Stress response
Cell wall/Cell division
Cell wall/Cell division
Virulence enzyme/toxin
Energy metabolism
Stress response
Cell wall/Cell division
Stress response
Stress response
Membrane transport
Energy metabolism
Membrane transport
Cell wall/Cell division
Stress response
Virulence adhesin
Energy metabolism
Energy metabolism
Membrane transport
Nucleic acid metabolism
Membrane transport
Virulence enzyme/toxin
Stress response
Membrane transport
Membrane transport
Virulence enzyme/toxin
Virulence regulator
Virulence biofilm
Membrane transport
Stress response
Virulence enzyme/toxin
Nucleic acid metabolism
Virulence regulator
Energy metabolism
Virulence enzyme/toxin
Nucleic acid metabolism
Energy metabolism
Virulence regulator
Cell wall/Cell division
Cell wall /Cell division
Cell wall/Cell division
Membrane transport
Membrane transport
Membrane transport
Membrane transport
Cell wall/Cell division
Membrane transport
Energy metabolism
Energy metabolism
Cell wall/Cell division
Stress response
Virulence enzyme/toxin
Virulence enzyme/toxin
Stress response
Virulence adhesin
Virulence enzyme/toxin
Energy metabolism
Membrane transport
Energy metabolism
Nucleic acid metabolism
Membrane transport
Stress response
Cell wall/Cell division
Energy metabolism
Stress response
Stress response
Virulence adhesin
Membrane transport
Membrane transport
Virulence regulator
Cell wall/Cell division
Energy metabolism
Membrane transport
Virulence enzyme/toxin
Cell wall/Cell division
Membrane transport
Cell wall/Cell division
Membrane transport
Membrane transport
Energy metabolism
Energy metabolism
Virulence enzyme/toxin
Virulence enzyme/toxin
Membrane transport
Stress response
Stress response
Membrane transport
Virulence enzyme/toxin
Virulence adhesin
Lipid metabolism
Membrane transport
Cell division protein
Molybdopterin biosynthesis MoeA protein, putative
Peptidase, M20/M25/M40 family
Penicillin-binding protein 3
Heat-inductible transcription repressor
Mn2+/Fe2+ transporter, NRAMP family
Cysteine protease
Drug resistance transporter
SsaA-like protein
TcaA protein, cell wall, teicoplanin and MRSA associated resistance
GGDEF Domain protein, c-di-GMP
membrane protein, similar to acyltransferase in N315
ABC transporter, permease/ATP-binding protein
ABC transporter, ATP-binding protein, not identified
Delta-hemolysin, RNAIII
Conserved hypothetical protein TIGR00370, homolog mazE E.coli
FtsL protein, putative
Glutamine synthetase repressor
Probable enterotoxin
Pyruvate dehydrogenase component, beta subunit
Squalene desaturase
UDP-N-acetylmuramate--alanine ligase
Anti-sigma factor B
Adaptations to atypical conditions
Potassium-transporting ATPase, A subunit
Respiratory nitrate reductase, gamma subunit
ABC transporter, ATP-binding protein, not identified
Cell division protein FtsA
Glucosamine-6-phosphate isomerase, putative, Csb33
Cell adhesion
Pyruvate dehydrogenase component, lipoamide dehydrogenase
Succinyl-CoA synthase
Homolog N315 sa 0825, type-I signal peptidase, spsA
Cmp-binding-factor 1 (mobile and extrachromosomal element functions)
ABC transporter, ATP-binding protein, not identified
Phospholipase, Phosphatidylinositol phosphodiesterase
Conserved protein
Heme-iron transport, heme-iron permease system
ABC transporter, ATP-binding protein
Exfoliatine Toxin production
Two-component response regulator systems
Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides
Copper translocating ATPase
Osmoprotectant proline transporter
Coagulase
Purine nucleoside phosphorylase
Transcriptional regulator, DeoR family, PTS system
Biosynthesis of cofactors, prosthetic groups, and carriers
Aerolysin/leukocidin family
Xanthine permease
Aspartate / ornithine carbamoyltransferase
Two-component response regulator systems
Cell division protein FtsZ
FemD protein, phosphoglucosamine mutase
Holin-like LrgB protein, murein hydrolase
Homolog N315 sa 0826 type-I signal peptidase 1B, spsB
Conserved hypothetical protein
ABC transporter, ATP-binding protein, not identified
Phosphate ABC transporter
N-acetylmuramoyl-L-alanine amidase
Potassium-transporting ATPase, C subunit
Alcool deshydrogenase, Zn containing
Delta-aminolevulinic acid dehydratase
Teichoic acid ABC transporter protein, putative
Homolog to staphyloxanthine biosynthesis proteins
Leukocidin
Thermonuclease
Chaperonine
Homolog to Pls, a putative MRSA adhesin
Cysteine protease SspC
Formate dehydrogenase, NAD-dependent
MmpL efflux pump putative
Isocitrate dehydrogenase, NADP-dependent
Adenylsuccinate lyase
Possible multi-drug efflux protein
drp35 protein, induced by detergents that perturb membrane integrity
Undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase
Ribose-phosphate pyrophosphokinase
hypothetical protein
Serine protease HtrA
Protein with cell sorting signals
Magnesium and cobalt transport protein CorA
Possible staphylobactin ABC transporter
transcriptional regulator, LysR family
Two-component response regulator systems
Transcriptional regulator NirR
ABC transporter, ATP-binding protein, not identified
Cellular processes: Toxin production and resistance
Transcriptional regulator, homolog peptide methionine sulfoxide reductase regulator MsrR
Phosphate ABC transporter
Response regulator
Membrane protein
Aminoacid permease
Uroporphyrinogen-III synthase HemD
Enoyl-CoA hydratase/isomerase family protein
Aureolysin, zinc metalloproteinase
Gamma hemolysin, component C
FeoB, Possible iron-tranport protein
Conserved domain protein
Sigma factor B regulator protein
Manganese ABC transporter, ATP-binding protein
Serine protease V8
Putative cell-surface protein, unknown ligand
Lipase/esterase
ABC transporter, ATP binding/permease with Fur box
-1,155378858
-1,154041159
-1,152830291
-1,148274663
-1,145810131
-1,141526507
-1,137845849
-1,131170203
-1,126525981
-1,125399717
-1,119435983
-1,117380663
-1,109815973
-1,106538915
-1,105256208
-1,102788025
-1,099762321
-1,098983521
-1,098939551
-1,095181631
-1,095082539
-1,092782922
-1,091214578
-1,090803584
-1,087411847
-1,086633388
-1,084961854
-1,084442746
-1,079173447
-1,078454614
-1,078257133
-1,076701208
-1,074700261
-1,074615504
-1,070706657
-1,069319769
-1,060242128
-1,05800892
-1,056143216
-1,055190699
-1,055004122
-1,054776924
-1,052270206
-1,046660509
-1,044740464
-1,044097442
-1,042779709
-1,04197603
-1,038085784
-1,036446371
-1,035365026
-1,029851824
-1,026784746
-1,021836199
-1,020516577
-1,020250282
-1,019000782
-1,018581883
-1,017094716
-1,016957085
-1,015784055
-1,011646805
-1,009182129
-1,008277401
-1,007118268
-1,006773567
-1,004825137
-1,004713741
-1,000671136
-0,99884969
-0,998814736
-0,996237494
-0,99422194
-0,992308737
-0,99026907
-0,986839296
-0,986137644
-0,986093492
-0,983642177
-0,983279917
-0,983094163
-0,982352891
-0,982297092
-0,982060913
-0,981742657
-0,978663393
-0,977835316
-0,977670076
-0,977357053
-0,977200022
-0,975699638
-0,973497384
-0,972583332
-0,968538018
-0,960944667
-0,957356672
-0,956318573
-0,955742087
-0,954826738
-0,954535176
-0,952743826
-0,951004845
-0,946542089
-0,945208408
-0,944790781
-0,232539035
-0,942980474
-0,846596146
-0,756625075
0,280069062
-0,189935182
-1,061718274
-0,431114173
1,234373938
-0,657359359
-0,284886989
-0,141703051
-0,528461199
-0,25488132
-3,162784462
-0,130763434
-0,838468985
-0,788601399
0,440227961
0,298374754
-0,405756695
-0,048382015
-0,422816977
0,027589959
-2,33715196
-0,124831259
-0,462714288
-0,767580958
-0,609535549
-0,471609817
-0,152755368
-0,143017236
-0,395108212
-0,750341923
-0,415126621
-0,81778672
-0,204965887
-0,249137201
-0,479052356
-0,626879935
0,444315544
-0,668160713
-0,673321728
-0,472584861
2,38003833
0,53102718
-0,122194871
-0,48387509
0,264432495
-4,412188978
-0,199509574
-0,131150572
-0,502283548
-0,709757377
-0,626151479
-0,107786935
-0,60189808
-0,286725966
0,967036501
0,070754225
-1,979233966
1,122692946
-0,608593522
-1,327670884
0,085546164
0,349642672
-0,698655039
0,25924396
-0,996701121
-0,56482643
-0,591206989
-0,645195874
0,332808915
-1,974674379
-0,808308747
-0,75885511
-0,436232916
-0,481878119
-0,167161916
-0,654387568
-0,840510969
-0,838902046
-0,559539909
-0,198417571
-1,123536138
0,141090995
-0,390232158
-0,263422646
-0,489618129
0,246220675
-1,144128005
-0,274302888
-0,562543677
-0,216460125
0,107575867
-0,549651197
1,06146974
-0,318323634
-0,378450992
-1,371437013
-6,289586915
-0,790955298
-0,576852991
-0,598108707
-0,387481364
539
718
798
1450
2408
2060
1013
415
638
31
505
2056
203
1933
1604
1640
1943
2395
1612
808
966
2509
50
2573
665
1739
1415
1611
118
2171
2194
1932
159
473
266
636
691
1072
2198
1719
2652
1164
2506
458
1263
1028
2416
2075
1423
97
1636
6
1329
1869
2657
944
666
247
551
856
2689
2017
2365
1806
422
1422
470
217
978
922
474
2580
2690
2511
2150
696
698
787
1352
472
2397
1416
2321
442
1040
1411
1864
2036
2419
504
460
1271
907
1353
264
1222
1270
1823
1180
842
1045
2024
2565
244
186
purR
sstC
arlS
alr
rsbV
Csb3
glkA
vraS
narG
pgi
fnbB
pls
copP
pho
sodA
hysA
sgtB
mvk
sirR
folD
budA1
hemA
clfB
sarT
xpt
sucD
sirC
dnaJ
gyrA
femC
splA
arcA
lrgA
ftsH
cflA
icaA
groES
icaD
fnbA
fmtB/sasB
tagB
tagD
Csb29
nasE
Csb28
femB
splF
hlgA
guaB
hsIU
ent2
rpoZ
hsIV
arsB
eno
agrD
feoA
scdA
Nucleic acid metabolism
Membrane transport
Membrane transport
Virulence regulator
Stress response
Cell wall/Cell division
Membrane transport
Stress response
Cell wall/Cell division
Cell wall/Cell division
Membrane transport
Stress response
Membrane transport
Stress response
Energy metabolism
Energy metabolism
Regulation
Energy metabolism
Membrane transport
Regulation
Energy metabolism
Virulence adhesin
Virulence adhesin
Membrane transport
Membrane transport
Stress response
Membrane transport
Regulation
Stress response
Membrane transport
Virulence enzyme/toxin
Cell wall/Cell division
Membrane transport
Virulence enzyme/toxin
Stress response
Cell wall/Cell division
Regulation
Energy metabolism
Energy metabolism
Energy metabolism
Virulence adhesin
Virulence adhesin
Virulence regulator
Nucleic acid metabolism
Energy metabolism
Virulence enzyme/toxin
Membrane transport
Virulence adhesin
Membrane transport
Membrane transport
Stress response
Nucleic acid metabolism
Cell wall/Cell division
Virulence enzyme/toxin
Energy metabolism
Energy metabolism
Membrane transport
Cell wall/Cell division
Cell wall/Cell division
Virulence adhesin
Virulence biofilm
Stress response
Stress response
Virulence adhesin
Membrane transport
Membrane transport
Virulence enzyme/toxin
Membrane transport
Membrane transport
Stress response
Virulence enzyme/toxin
Stress response
Virulence biofilm
Virulence adhesin
Virulence adhesin
Cell wall/Cell division
Cell wall/Cell division
Stress response
Membrane transport
Virulence enzyme/toxin
Energy metabolism
Membrane transport
Stress response
Virulence enzyme/toxin
Membrane transport
Cell wall/Cell division
Virulence enzyme/toxin
Membrane transport
Virulence enzyme/toxin
Membrane transport
Nucleic acid metabolism
Stress response
Virulence enzyme/toxin
Membrane transport
Membrane transport
Nucleic acid metabolism
Stress response
Membrane transport
Virulence enzyme/toxin
Energy metabolism
Membrane transport
Virulence regulator
Membrane transport
Cell wall/Cell division
Membrane transport
Pur operon repressor
ABC transporter, ATP-binding protein, not identified
Possible ferrichrome ABC transporter ATP-binding protein
Sensor histidine kinase
Lipoprotein, putative, Homolog mazE E.coli
Alanine racemase
Magnesium transporter
Dyp-type peroxidase family protein
Phosphomevalonate kinase
Glycerophosphoryl diester phosphodiesterase, putative
ABC transporter, ATP-binding protein, not identified
Anti-anti-sigma factor
Possible iron-binding protein
ThiJ/PfpI family protein, Csb3
Glucokinase
Oxygen-independent coproporphyrinogen III oxidase
Two-component response regulator systems
Nitrate reductase
ABC transporter, permease protein
membrane protein putative, related to c-di-GMP
Glucose-6-phosphate isomerase
Fibronectin-binding protein, FNBPs
Methicillin resistant surface protein
Copper-ion-binding protein
Possible ferrichrome ABC transporter permease
Two-component response regulator systems
ABC transporter, ATP-binding protein, not identified
Transcription regulator Fur family homolog
Superoxide dismutase
Aerobactin biosynthesis protein, IucA/IucC family
Hyaluronidase
Transglycosylase domain protein
ABC transporter, permease
Exotoxin 5, putative
Hypothetical protein
mevalonate kinase
Iron dependent repressor
Methylenetetrahydrofolate dehydrogenase/cyclohydrolase
Alpha-acetolactate decarboxylase
Glutamyl-tRNA reductase
Ser-Asp rich FNBP
Protein with cell wall sorting signal, fibrinogen-binding protein
Staphylococcal accessory regulator T
Xanthine phosphoribosyltransferase
Succinyl-CoA synthase
Hypothetical protein, probable serine protease
Cation efflux familiy protein
Proteins with cell sorting signals
Phosphate ABC transporter
Possible staphylobactin ABC transporter
Protein folding and stabilization
DNA gyrase, A subunit
Biosynthesis of murein sacculus and peptidoglycan
Protease
Arginine deiminase
NADH dehydrogenase
Possible ferrichrome ABC transporter permease
Holin-like LrgA protein, murein hydrolase
Cell-division initiation protein, putative
Clumping factor A
Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides
Chaperonine
Conserved hypothetical protein
Proteins with cell sorting signals
ABC transporter, ATP-binding protein, not identified
Phosphate ABC transporter
Exotoxin, putative
ABC transporter, ATP-binding protein, not identified
Membrane protein putative
Conserved hypothetical protein
Exotoxin 4, putative
Hypothetical protein
Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides
Fibronectin-binding protein A
FmtB, proteins with cell sorting signals, associated to MRSA
Teichoic acid biosynthesis protein B
Cholinephosphate cytidylyltransferase, putative
Conserved protein, Csb29
ABC transporter, ATP-binding protein, not identified
Exotoxin, putative
Nitrate reductase possible
ABC transporter, ATP-binding protein, not identified
Oxidoreductase dehydrogenase/reductase, Csb28
Possible exotoxin
ABC transporter, ATP-binding protein, not identified
Biosynthesis of murein sacculus and peptidoglycan
Probable serine protease
ABC transporter, ATP-binding protein, not identified
Gamma-hemolysin, component A
ABC transporter, ATP-binding protein, not identified
Inosine-5-monophosphate dehydrogenase
Heat shock protein HslVU
Enterotoxin
ABC transporter, ATP-binding protein, not identified
ABC transporter, ATP-binding protein, not identified
DNA-directed RNA polymerase, omega subunit
Heat shock protein HslV
Arsenical pump membrane protein
Exotoxin 3
Enolase
Possible ferrichrome/Iron ABC transp. ATP-binding
Accessory gene regulator protein D
FeoA domain protein
Cell division and morphogenesis-related protein
ABC transporter, ATP-binding protein, not identified
-0,943206542
-0,943103062
-0,942560914
-0,941664906
-0,938503954
-0,937938769
-0,936239218
-0,935914836
-0,935847213
-0,935739178
-0,935353765
-0,93417832
-0,933851786
-0,928053783
-0,927100342
-0,927026923
-0,92616882
-0,926014218
-0,925613496
-0,925360957
-0,922687183
-0,92252976
-0,920535838
-0,920522342
-0,92014351
-0,919055966
-0,91848836
-0,91725191
-0,917145401
-0,916490194
-0,915049447
-0,912530921
-0,912372608
-0,912287495
-0,911800931
-0,909751895
-0,909561018
-0,909418847
-0,909314822
-0,908633068
-0,907974879
-0,90737888
-0,90722258
-0,906945603
-0,906894932
-0,90148633
-0,89914569
-0,898599994
-0,897450712
-0,893922548
-0,891120101
-0,890050408
-0,889083262
-0,886436759
-0,885214346
-0,884903449
-0,883922982
-0,883389547
-0,882921168
-0,881845259
-0,88068519
-0,878464038
-0,874680916
-0,874518035
-0,870716298
-0,865475702
-0,865463273
-0,857755052
-0,857686158
-0,856916759
-0,855032359
-0,850852564
-0,850025692
-0,843984307
-0,841417048
-0,837470195
-0,836930643
-0,836830937
-0,835867255
-0,832656056
-0,824953414
-0,823811792
-0,823468007
-0,819300791
-0,817806848
-0,8158206
-0,808175076
-0,804051787
-0,804043559
-0,803727008
-0,803407808
-0,803065041
-0,80179727
-0,800420926
-0,797668718
-0,791217995
-0,790529428
-0,790005898
-0,784577079
-0,784383846
-0,783604891
-0,78206127
-0,778001598
-0,777811457
-0,771905967
-0,471992655
-0,558095287
-0,012882906
-1,504898743
-1,131541865
-1,184134172
-0,764846957
-0,444192227
-0,279172678
-0,306020306
-0,600585053
-0,310575981
-0,655407595
-0,40580854
-0,09478348
-0,332159724
-0,620808027
0,285723525
-1,17526222
-0,478838979
-0,80252837
0,704385477
-0,423932838
-0,601541761
-1,069459833
-0,363601453
-0,185669479
-0,083744712
-1,17035484
-0,531344197
-0,266496225
-0,299870355
-0,391238945
-0,289822481
-0,355388873
0,125679077
-0,226882444
-1,197035114
-0,457846131
0,090519796
-0,744246872
0,959801133
-0,344876927
-4,945077803
0,000591939
-1,25436124
0,243143703
-0,257376636
-0,089906991
0,040848432
-0,124611712
-0,021619811
-0,852260145
-0,33098825
0,004037635
-0,252300069
-1,077052165
-0,490228774
-0,365075662
0,247179289
-0,360292956
0,28816261
-0,169352827
-0,829089786
-0,452710662
-0,079960744
-0,283076649
-0,578146766
-0,653529842
-0,434718902
-0,535679779
0,127470741
-0,249587101
-0,244323048
-0,405383007
-0,412496594
-0,678837573
0,212835513
-0,392595363
-0,385059426
-0,008254679
-0,333608743
-0,628858711
-1,063035687
-0,369254199
-0,197605097
-0,357876873
-0,982220764
1,363990325
-0,454821648
-1,740076261
0,096424683
-0,261649507
-0,515260978
-0,35113764
-1,997058748
0,080987877
-0,487158134
-0,320329591
-0,02838933
-0,543279555
-0,268434416
-0,4459861
-0,146122213
-0,358414874
155
2375
979
369
185
1881
1717
2073
317
1390
506
2253
941
1921
2398
1056
478
693
1714
2002
1461
2379
2010
2144
2426
697
2256
1327
220
797
1023
1417
2683
2599
884
2003
841
1459
839
184
1868
2189
1169
723
1867
1451
706
1942
1781
779
705
1880
1721
1618
1441
1052
1929
2166
2168
2531
1178
921
1453
840
2138
1168
882
461
2074
2692
2385
1389
2136
2554
418
1146
977
1951
1221
1866
567
976
2563
1745
1143
1893
2165
1410
1905
2173
556
2211
2092
2584
1179
2549
301
1397
780
1144
1145
2116
796
2553
99
clpB
clpP
lukE
hemC
murF
geh
parC
femX
bcp
nirB
sspB
tagA
hemL
map
dfrA
Csb19
bioB
tagX
hml
sstB
murE
hlb
pgm
msrA
pgk
splB
ssaA
splC
arlR
fhuG
vraR
sasI/isdH
fhuB
lukD
clpX
rpoD
menD
htsB
murG
tpiA
fib/efb
guaA
ddlA
icaC
parE
Csb9
cidB
isdG
frpA
gmk
splD
ctsR
clp
pyk
isdE
htsA
femA
vraR
asp23
murA
isaA
msrA
recQ
isdF
srtB
murZ
sstA
cidC
sirA
Membrane transport
Membrane transport
Stress response
Stress response
Membrane transport
Virulence enzyme/toxin
Energy metabolism
Cell wall/Cell division
Virulence enzyme/toxin
Nucleic acid metabolism
Membrane transport
Cell wall/Cell division
Energy metabolism
Iron metabolism
Energy metabolism
Virulence enzyme/toxin
Virulence enzyme/toxin
Cell wall/Cell division
Energy metabolism
Stress response
Nucleic acid metabolism
Stress response
Membrane transport
Membrane transport
Energy metabolism
Cell wall/Cell division
Stress response
Stress response
Energy metabolism
Membrane transport
Cell wall/Cell division
Membrane transport
Cell wall /Cell division
Membrane transport
Membrane transport
Virulence enzyme/toxin
Energy metabolism
Cell wall/Cell division
Energy metabolism
Membrane transport
Virulence enzyme/toxin
Regulation
Virulence adhesin
Cell wall/Cell division
Virulence enzyme/toxin
Virulence regulator
Iron metabolism
Regulation
Membrane transport
Membrane transport
Membrane transport
Virulence enzyme/toxin
Stress response
Regulation
Membrane transport
Energy metabolism
Membrane transport
Membrane transport
Membrane transport
Stress response
Virulence enzyme/toxin
Virulence enzyme/toxin
Cell wall/Cell division
Energy metabolism
Membrane transport
Virulence adhesin
Membrane transport
Nucleic acid metabolism
Cell wall/Cell division
Virulence biofilm
Stress response
Nucleic acid metabolism
Stress response
Cell wall/Cell division
Membrane transport
Membrane transport
Membrane transport
Cell wall/Cell division
Energy metabolism
Virulence enzyme/toxin
Stress response
Membrane transport
Stress response
Energy metabolism
Membrane transport
Membrane transport
Membrane transport
Cell wall/Cell division
Regulation
Stress response
Stress Response
Membrane transport
Cell wall/Cell division
Virulence adhesin
Virulence enzyme/toxin
Lipid metabolism
Energy metabolism
Cell wall/Cell division
Nucleic acid metabolism
Membrane transport
Virulence adhesin
Cell wall/Cell division
Membrane transport
Cell wall/Cell division
Iron metabolism
Cation efflux familiy protein
Membrane transporter
Protease SigA-dependant stress genes / TIGR 2007= ATP-dependent Clp protease, ATP-binding subunit ClpB
Protease SigA-dependant stress genes / TIGR 2007=Prophage L54a, Clp protease, putative
ABC transporter, ATP-binding protein, not identified
Leukotoxin
Porphobilinogen deaminase
UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase
Lipase, glycerol ester hydrolase
DNA topoisomerase IV, A subunit
ABC transporter, ATP-binding protein, not identified
FemX protein, peptidyl transferase, putative FmhB
NADH dehydrogenase
Bacterioferritin comigratory protein
Nitrate reductase, large subunit
Cysteine protease SspB
Exotoxin 3, putative
UDP-N-acetyl-D-mannosamine transferase
Glutamate-1-semialdehyde-2,1-aminomutase
Protein modification and repair
Dihydrofolate reductase
Conserved protein, Csb19 (biosynthesis/metabolism-associated gene in Moisan)
Iron-compound ABC transporter, subst. binding
ABC transporter, ATP-binding protein, not identified
Biotin synthetase
Teichoic acid biosynthesis protein X
Transcriptional regulator, MarR family
Aluminum resistance protein
Flavohemoprotein
Possible ferrichrome ABC transporter permease
UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase
ABC transporter, ATP-binding protein, not identified
Peptide methionine sulfoxide reductase, putative
Conserved domain protein
ABC transporter, ATP-binding protein, not identified
beta hemolysin
Phosphoglycerate mutase (mal, gal to G1-P to G6-P)
Methionine sulfoxide reductase
Phosphoglycerate kinase
ABC transporter, ATP-binding protein, not identified
Probable serine protease
Transcriptionnal regulator, Sir 2 family
Fibrinogen-binding protein
Homolog to streptococcal secretory antigen A, LysM domain and Antigenic surface protein
Probable serine protease
DNA-binding response regulator
Ferrichrome transport permease
DNA-binding response regulator, VraR
Cell wall surface anchor family protein, harA N315 homolog
ABC transporter, ATP-binding protein, not identified
Ferrichrome transport permease
Leukotoxin
ATP-dependent Clp protease, ATP-binding subunit ClpX
RNA polymerase sigma factor, homolog sigA
Conserved hypothetical protein, similar to tellurite resistance prot
Carboxylate synthase
ABC transporter, ATP-binding protein, not identified
Possible heme-iron transport system, ABC transporter permease
Conserved hypothetical protein
Transcriptional regulator, MarR family
Exotoxin 1
Probable hemolysin
UDP-GlucNac-MurNac-(pentapeptide) PP-C55-GlucNac transferase
Triosephosphate isomerase
Cation efflux familiy protein
Fibrinogen-binding protein
ABC transporter, ATP-binding protein, not identified
GMP synthetase
D-alanine-D-alanine ligase
Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides
Heat shock protein, Hsp20 family
DNA topoisomerase IV, B subunit
Conserved protein, Csb9 (biosynthesis/metabolism-associated genes in Moisan)
Membrane protein, putative, linked to LrgAB
MttA/Hcf 106 family protein with Fur box
Heme-iron transport, cytoplasmic protein
Fur-regulated protein A, possible membrane protein (Allard frp operon, Morissey 2002 IAI)
Mur ligase family protein
guanylate kinase
Probable serine protease
Transcriptional regulator
Hydrolase, haloacid dehalogenase like familly
ATP-dependant protease
Pyruvate kinase
Heme-iron transport, heme-iron permease system
ABC transporter, ATP-binding protein, not identified
Possible heme-iron transport system, ABC transporter permease
Biosynthesis of murein sacculus and peptidoglycan
DNA-binding response regulator, VraR
Alkaline shock protein 23
Chaperonine, Hsp33 homolog
ABC transporter, ATP-binding protein
UDP-N-acetylglucosamine 1-carboxyvinyltransferase
Immunodominant protein A, lytic transglycosylase
Exotoxin 2
Esterase, putative
Formate / nitrite transporter protein
Methionine sulfoxide reductase
ATP-dependent DNA helicase
Heme-iron transport, heme-iron permease system
Sortase B
UDP-N-acetylglucosamine 1-carboxyvinyltransferase
Possible ferrichrome ABC transporter permease
Pyruvate oxidase, linked to LrgAB
Possible staphylobactin ABC transporter
-0,768143394
-0,766861519
-0,764265876
-0,762749026
-0,756513723
-0,748803699
-0,748542827
-0,739690799
-0,725724572
-0,722002876
-0,715387049
-0,71490505
-0,71086179
-0,703987286
-0,699084988
-0,693435424
-0,692515714
-0,690842417
-0,689428109
-0,68838679
-0,687400348
-0,683059458
-0,682411402
-0,679458061
-0,67939942
-0,679382568
-0,672568715
-0,672217932
-0,671439084
-0,662487706
-0,662435175
-0,660888497
-0,65820488
-0,654324976
-0,65125332
-0,646444332
-0,646020719
-0,640561017
-0,635067291
-0,631696813
-0,627785234
-0,625914494
-0,610822735
-0,609791654
-0,606036692
-0,59988448
-0,596443362
-0,596350395
-0,5962317
-0,593874764
-0,585925486
-0,581911764
-0,571372073
-0,567150787
-0,564374034
-0,563448791
-0,560001835
-0,558845823
-0,553427574
-0,548557225
-0,546812966
-0,546000271
-0,544131545
-0,542117397
-0,537130955
-0,536728793
-0,531532615
-0,521961389
-0,512642298
-0,501443227
-0,501300337
-0,498343732
-0,490032201
-0,484544005
-0,481746482
-0,481485171
-0,475642463
-0,468031201
-0,465149598
-0,440698698
-0,432562533
-0,425857928
-0,420092125
-0,419470966
-0,416225977
-0,414214275
-0,410047718
-0,404265772
-0,40389511
-0,402682756
-0,395398997
-0,395212014
-0,394821319
-0,393106072
-0,391951846
-0,387754389
-0,381274143
-0,37977698
-0,379563316
-0,37928743
-0,372220844
-0,364536938
-0,330102334
-0,326950201
-0,324193739
-0,380517377
-0,670224644
0,879255482
-0,248117208
-0,344838803
-0,31096108
0,036186801
-0,204015309
-1,832117496
0,188981176
-0,719865502
-1,678047543
-0,349664431
-0,757401463
-0,132129483
-0,861435483
0,472855402
-1,376789308
-0,470425232
-0,328957716
-0,272902112
-0,226112211
-0,27455695
-0,344631569
0,257238378
-0,838534669
-0,734782772
-0,028873252
-0,266022815
0,071398809
-1,265048124
-0,406760917
-0,754252227
-0,530327719
-0,898640558
-0,2184672
0,22992182
-0,017199382
0,227281434
-0,453737182
-0,336740498
-0,277545059
0,258617461
-0,198324808
-0,41878155
-1,521129989
-0,477577942
-0,44371612
-0,708029618
-0,636593048
-0,510911908
-0,433423112
0,203020638
-0,149912775
-0,112167132
-0,042229077
-0,69396101
-0,321298883
-0,761001575
-0,391155341
-0,369603961
-0,202288294
-0,901223832
0,343197672
-1,410895793
0,273391486
-1,143280387
-1,231757361
-0,797704001
-1,004588498
-0,606156165
-0,330256468
-0,394333239
-0,885682767
-0,937958972
-0,874986936
-0,805343957
-0,374642707
-2,094146605
-0,49022983
-0,035488553
-0,575828747
0,050904427
-0,670363028
-1,15506456
-0,547092356
-0,323735069
-0,346680934
-0,678291588
0,084054844
-0,354103121
-1,099195772
-0,106897565
-0,914943217
-0,330865902
-0,515575362
1,826110802
-1,074154225
-0,407813618
-1,156200689
-1,051411791
-0,532242656
-0,309802519
0,819756111
-0,805022489
2555
1887
507
2169
107
2210
1511
1687
104
106
1984
1141
1062
617
105
857
1161
108
694
2277
1889
2114
1919
102
101
1888
222
823
2369
1138
1746
2088
2534
2117
161
204
2167
838
453
2170
100
263
829
914
824
704
205
1368
2539
1541
1140
1952
451
452
cidR
hemY
sbnH
gerCB
lytH
sbnE
sbnG
aldA
isdC
atl
Csb4
sbnF
murI
sbnI
tagH
fhuD2
hemE
ald/Csb24
sbnC
sbnB
hemH
ldh
uvrB
isdB
pfk
sceD
fba
pflB
htsC
gapA1
sbnA
lytM
trxB
uvrA
fhuA
pflA
katA
srtA
isdA
ahpF
ahpC
Cell wall/Cell division
Energy metabolism
Membrane transport
Membrane transport
Membrane transport
Membrane transport
Stress response
Cell wall/Cell division
Membrane transport
Membrane transport
Energy metabolism
Membrane transport
Cell wall/Cell division
Energy metabolism
Membrane transport
Virulence enzyme/toxin
Cell wall/Cell division
Membrane transport
Cell wall/Cell division
Membrane transport
Energy metabolism
Stress response
Regulation
Membrane transport
Membrane transport
Energy metabolism
Energy metabolism
Stress response
Stress response
Membrane transport
Energy metabolism
Virulence enzyme/toxin
Energy metabolism
Energy metabolism
Membrane transport
Energy metabolism
Membrane transport
Energy metabolism
Energy metabolism
Membrane transport
Membrane transport
Cell wall/Cell division
Stress response
Membrane transport
Stress response
Membrane transport
Energy metabolism
Stress response
Virulence adhesin
Regulation
Membrane transport
Stress response
Stress response
Stress response
Transcriptional regulator, LysR family, putative, linked to LrgAB
Porphyrin biosynthesis
LysM domain protein
Aerobactin biosynthesis protein, IucA/IucC family
Staphylobactin biosynthesis
ABC transporter, ATP-binding protein, not identified
Methlytransferase, UbiE/COQ5 family
N-acetylmuramoyl-L-alanine amidase, putative
Staphylobactin biosynthesis
Staphylobactin biosynthesis
Aldehyde dehydrogenase
Heme-iron transport, NPQTN cell wall surface anchored protein
Bifunctionnal autolysin
Hexulose-6P synthase, Csb4 (biosynthesis/metabolism in Moisan et al)
Staphylobactin biosynthesis
Staphylocoagulase, putative
Glutamate racemase
Staphylobactin biosynthesis
Teichoic acid ABC transporter protein, putative
Ferrichrome transport, receptor
Porphyrin biosynthesis
Aldehyde dehydrogenase, Csb24 (biosynthesis/metabolism-associated genes in Moisan)
Transcription regulator Fur family homolog
Staphylobactin biosynthesis
Staphylobactin biosynthesis
Porphyrin biosynthesis
L-lactate deshydrogenase
Excinuclease ABC, B subunit
Disulfide reductase with Fur box
Heme-iron transport, LPXTG cell wall surface anchored protein
Phosphofructokinase
Exotoxin, SceD like
NAD(P)H-flavin oxidoreductase
Fructose-bisphosphate aldolase, class II
Conserved hypothetical protein with Fur box
Formate acetyltransferase
Possible heme-iron transport system, ABC transporter, ATP-binding
Glyceraldehyde 3P deshydrogenase
NAD(P)H-flavin oxidoreductase
Putative transporter with Fur box
Staphylobactin biosynthesis
Peptidoglycan hydrolase
Thioredoxin reductase (TIGR function class= electron transport, nitrofurantoin resistance)
ABC transporter, ATP-binding protein, not identified
Excinuclease ABC, A subunit
Ferrichrome transport ATP-binding protein
Formate-lyase activating enzyme
Catalase SigA dependant gene / TIGR 2007=Catalase
Sortase A
Transcription regulator Fur family homolog
Heme-iron transport, LPXTG cell wall surface anchored protein
Possible ferritin
Alkyl hydroperoxide reductase, subunit F
Alkyl hydroperoxide reductase, subunit C
-0,313110991
-0,307274001
-0,298971801
-0,281471681
-0,24064555
-0,239605945
-0,230354965
-0,224896241
-0,211359697
-0,210855278
-0,208133864
-0,203222064
-0,182867036
-0,146422417
-0,119042594
-0,099920266
-0,052965657
-0,030064786
-0,027145434
-0,023880714
-0,008349284
-0,003087953
0,009741377
0,013211987
0,025775588
0,048484607
0,06538663
0,075490791
0,080741937
0,099114147
0,104578944
0,113578697
0,137619491
0,154406674
0,154967622
0,160268762
0,189299191
0,195618132
0,206161855
0,276822384
0,305522302
0,329512894
0,339969956
0,449018214
0,465082723
0,478879651
0,544383132
0,622188191
0,62989621
0,650908345
0,945305109
1,245132061
2,421742835
2,781390482
-0,920923172
0,029810662
-0,500451514
-0,4828043
-0,121733613
-0,847719808
-0,390197058
-1,240728036
-0,218290965
-0,196577033
-0,583157583
-1,186225189
-0,023445846
-0,383670442
-0,040558377
0,810263105
-1,219944185
-0,169398254
-1,647250143
-0,329746023
-0,10551987
0,360060297
0,116783865
0,257270993
-0,250521798
0,171279651
5,44162314
1,314080714
-0,271516781
-0,808806945
-1,287033354
2,205689824
-0,545136306
-0,08453544
-1,513413045
2,23013755
0,456378173
0,562624807
0,117231162
-0,643914351
-0,31931345
1,314461326
-0,373023245
1,049368599
1,517744316
-0,019215495
1,513805381
0,096512641
-0,810575454
0,47767602
-1,742332797
0,471733164
1,980538629
1,87988606