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Mycobactin MbtA regulation!
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Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis*.
Olivia Vergnolle,1 Hua Xu,1 JoAnn M. Tufariello,2 Lorenza Favrot,1 Adel A. Malek,2 William R.
Jacobs Jr2 and John S. Blanchard1
1Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
New York 10461, USA
2Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein
College of Medicine, Bronx, New York 10461, USA
*Running title: Mycobactin MbtA regulation
To whom correspondence should be addressed: John S. Blanchard, Department of Biochemistry, Albert
Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA, Tel.: +001 718
430 3096; E-mail: john.blanchard@einstein.yu.edu
Keyword: MbtA, acetylation, structure, mycobactin, Mycobacterium tuberculosis
Iron is an essential element for life, but its
soluble form is scarce in the environment and is
rarer in the human body. Mycobacterium
tuberculosis (Mtb) produces two aryl-capped
siderophores, mycobactin (MBT) and
carboxymycobactin (cMBT), to chelate
intracellular iron. The adenylating enzyme
MbtA catalyzes the first step of mycobactin
biosynthesis in two half-reactions: activation of
the salicylic acid as an acyl-adenylate and
ligation onto the acyl carrier protein (ACP)
domain of MbtB to form covalently salicylated
MbtB-ACP. We report the first apo-MbtA
structure from Mycobacterium smegmatis at 2.3
Å. We demonstrate here that MbtA activity can
be reversibly, post-translationally regulated by
acetylation. Indeed the mycobacterial protein
acetyltransferase (Pat), Rv0998, specifically
acetylates MbtA on lysine 546, in a cAMP-
dependent manner, leading to enzyme
inhibition. MbtA acetylation can be reversed by
the NAD+-dependant deacetyltransferase,
Rv1151c (DAc). Deletion of Pat and DAc genes
in Mtb revealed distinct phenotypes for strains
lacking one or the other genes at low pH and
limiting iron conditions. This study establishes
a direct connection between the reversible
acetylation system Pat/DAc and the ability of
Mtb to adapt in limited iron conditions, which is
critical for mycobacterial infection.
Iron is the most abundant element in the
earth’s core and represent 5% of the earth crust
(1). However, because ferric iron, Fe(III), is
poorly soluble in water at neutral pH, its biological
availability is scarce (10-18 M in water) (2). Iron is
a vital nutrient for most prokaryotes and
eukaryotes with a notable exception among well
studied pathogenic organisms being the Lyme
disease pathogen, Borrelia burgdorferi, which
uses manganese (3). Iron is required for DNA
replication, oxygen transport, energy generation,
enzymatic redox reactions and oxidative stress
protection. Due to the scarcity of free iron,
essentially all living species have evolved ways to
acquire and store iron and regulate iron levels. In
humans, the majority of iron is found in the
porphyrin ring of heme proteins, in iron/sulfur-
containing enzymes or sequestered in the ferritin
protein. To prevent pathogen colonization, humans
deplete free iron availability to a concentration of
10-24 M in serum using the transferrin protein (4)
and macrophages decrease the expression of
ferritin and the transferrin receptor to keep
intracellular macrophage iron levels low (5). To
circumvent host protection mechanisms, the
intracellular pathogen Mycobacterium tuberculosis
(Mtb) has fine-tuned an effective iron-chelating
arsenal during its 70,000 year old cohabitation
with humans (6). Mtb acquires iron in two distinct
ways: a high-affinity heme acquisition system
(7,8) and via a siderophore-based system (9-11).
Mtb synthesizes two related aryl-capped
polyketide-polypeptide siderophores, called
mycobactin (MBT) and carboxymycobactin
(cMBT) (Fig. 1A) (12). These siderophores differ
only in the composition of their aliphatic tails,
http://www.jbc.org/cgi/doi/10.1074/jbc.M116.744532The latest version is at
JBC Papers in Press. Published on August 26, 2016 as Manuscript M116.744532
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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which determine the localization of the
siderophore: long-chain fatty acylated MBT is
membrane-associated while cMBT contains short-
chain dicarboxy-fatty acids and is more water-
soluble and is secreted by the pathogen (13,14).
Two MBT gene clusters, Mbt-1 and Mbt-2 (Fig.
1B), are composed of a mixture of nonribosomal
peptide synthetase and polyketide synthase
enzymes (15). Three specific enzymes, MbtA,
MbtM (also known as FadD33) and MbtK are
essential for mature mycobactin biosynthesis (16).
MbtA catalyzes the first step of MBT core
synthesis by activating salicylic acid as an
acyladenylate before subsequent ligation to the
pantothenyl group of the acyl carrier protein
domain of MbtB (Fig. 1C) (12). FadD33 activates
the long chain fatty acid before its transfer onto
the central N-hydroxy lysine residue of the MBT
core (16). MbtK is a GNAT N-acetyltransferase
that transfers the long chain fatty acid onto the
mycobactin core (16).
Iron-dependent transcriptional regulators,
IdeR and HupB, control the transcription level of
genes involved in iron metabolism including Mbt-
1 and Mbt-2 in order to avoid excessive iron
uptake, storage and toxic overload in the bacterial
cell (17,18). Another level of MBT regulation was
recently characterized as a post-translational
acetylation of FadD33 by the protein lysine
acetyltransferase (Pat) in Mycobacterium
smegmatis (Msmeg) (19). Pat acetylates and
inactivates FadD33 in a cAMP-dependent manner,
which is then reversed by a NAD+-dependent
Sirtuin-like deacetylase, DAc1, to restore FadD33
activity. Pat has been structurally characterized
and consists of an N-terminal cAMP-binding
domain fused to a C-terminal GCN5-related N-
acetyl-transferase domain (GNAT) (20,21). The
N-terminal domain allows Pat to be activated by
the second messenger, cAMP, produced by the
adenylate cyclase (AC) enzymes. The Mtb genome
encodes a total of 16 ACs, which are sensitive to
diverse environmental stimuli, including pH,
hypoxia, fatty acids and carbon dioxide level (22).
This complex response system allows
mycobacteria to rapidly adapt to external changes
by relaying this information, via a specific cAMP
pathway, to downstream effectors.
In this study, we demonstrate that the first
committed step in mycobactin biosynthesis,
catalyzed by MbtA, can also be reversibly post-
translationally acetylated by Pat with the loss of its
enzymatic activity, and reactivated by DAc
through deacetylation. Deletion of Pat and DAc
genes in Mtb highlights the importance of those
specific two genes for normal MBT production
during iron starvation.
RESULTS
Cloning, Expression, and Purification of MbtA. To
avoid solubility problems, Mtb and Msmeg N-
terminal-His6-tagged MbtA were expressed at low
temperature. Heterologous expression in E. coli
gave good quantities of soluble MbtA. After
purification by nickel affinity chromatography an
apparent molecular weight of ~59 kDa was
observed by SDS-PAGE, which is consistent with
the predicted 58983 Da and 59280 Da molecular
weight for Msmeg and Mtb respectively. Mtb and
Msmeg MbtA share 69.2 % sequence identity and
85.0 % sequence similarity.
Cloning, Expression, and Purification of MbtB.
The Mtb ACP domain of MbtB module was
expressed in E. coli but was mostly insoluble.
Using denaturing conditions, small quantities of
soluble apo-MbtB-ACP were obtained after nickel
affinity chromatography. As assessed by SDS-
PAGE, apo-MbtB-ACP displayed an apparent
molecular mass of ∼11 kDa, consistent with the
molecular weight of 11670 Da calculated from the
amino acid sequence. For the subsequent activity
assay with MbtA, apo-MbtB-ACP is required to be
phosphopantetheinylated (holo-MbtB-ACP form).
Sfp, a phosphopantetheinyl transferase from
Bacillus subtilis (B. subtilis) was used to
covalently transfer the 4’-phosphopantetheinyl
group from coenzyme A onto apo-MbtB-ACP as
described in the Materials and Methods section.
The conversion of apo-MbtB-ACP to holo-MbtB-
ACP was confirmed by addition of 340 Da moiety,
corresponding to the phosphopantetheine arm by
Fourier transform mass spectral analysis (data not
shown).
Specific acetylation of MbtA by protein lysine
acetyltransferase. Recently, protein lysine
acetyltransferase (Pat) has been identified as a
regulator of one of the later step of mycobactin
biosynthesis via post-translational modification of
fatty acyl-AMP ligase FadD33 (19). MbtA and
FadD33 both catalyze an adenylation reaction
followed by thioesterification of the substrate onto
an ACP. FadD33 is acetylated by Pat on K511 and
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amino acid sequence comparison of MbtA and
FadD33 highlights a conserved lysine residue,
K546, in MbtA (Fig. 2A). Moreover like FadD33-
K511, MbtA-K546 is also flanked by a preceding
glycine as well as two nearby downstream basic
residues, which are believed to lower the steric
hindrance between Pat and MbtA and reduce the
pKa value of the NH2 group of K546 respectively
(Fig. 2A). Based on these observations we probed
MbtA as a potential substrate for Pat. For this
experiment Msmeg Pat (MSMEG_5458) was used
instead of Mtb Pat (Rv0998) since protein
refolding issues occurred after protein purification
therefore affecting Mtb Pat activity. Western-blot
analysis using anti-acetyllysine antibody shows
that MbtA is acetylated by Pat in the presence of
acetyl-CoA and cAMP (Fig. 2B, lane 2). Negative
controls without Pat or acetyl-CoA show no MbtA
acetylation (Fig. 2B, lanes 3 and 4). Only in the
negative control without cAMP, a light MbtA
acetylation is observed (Fig. 2B, lane 5) likely due
to co-purification of Pat with some bound cAMP
as noted previously (23). Msmeg Pat also
acetylates Mtb MbtA in the same manner (data not
shown). To determine the site(s) of acetylation we
created a single amino acid change in MbtA,
changing K546 to an alanine. K546 appears to be
the main acetylation site since the K546A mutant
completely loses the ability to be acetylated by Pat
by western-blot (Fig.2B, lane 1). To confirm this
unique acetylation site, in vitro acetylated MbtA
was analyzed by mass spectrometry. Good
sequence coverage (85%) allowed the
identification of K546 as the single acetylation site
(Fig. 2C) and confirms that Pat acetylates MbtA
on K546.
Effect of Acetylation on MbtA Activity. MbtA
enzymatic activity was followed by monitoring the
formation of AMP in the presence of 2,3-
dihydroxybenzoate and acceptor holo-MbtB-ACP.
To test acetylation effects, the activity of Msmeg
MbtA was monitored overtime in the presence of
Msmeg Pat, acetyl-CoA and cAMP. After 5 hrs of
incubation, Msmeg MbtA activity was reduced by
80 % whereas MbtA activity without either acetyl-
CoA or Pat remained unchanged (Fig. 3A). The
gradual loss of activity is directly mediated by
MbtA acetylation in a Pat- and acetylCoA-
dependent manner. Using the same Mtb MbtA and
Msmeg Pat ratio as used previously for Msmeg
MbtA, 12 hrs of incubation were necessary to
achieve complete Mtb MbtA inactivation (data not
shown). For all subsequent experiments MbtAs
were incubated overnight with Msmeg Pat to
insure full inactivation. The Mtb genome encodes
one known sirtuin-like deacetylase DAc
(Rv1151c) in contrast to the Msmeg genome,
which contains two sirtuin-like deacetylases
named DAc1 (Msmeg_5175) and DAc2
(Msmeg_4620). DAc and DAc1 are very similar
and were previously shown to deacetylate
acetylated forms of acetyl-CoA synthetase (ACS)
and FadD33 (19,23). Western-blot analysis of
acetylated Mtb and Msmeg MbtA after overnight
incubation with DAc deacetylase demonstrates
that both MbtAs are deacetylated by DAc (Fig.
4A, lane 2 and 4B, lane 3). To check if
deacetylation restores MbtA activity, acetylated
MbtA was incubated with DAc and NAD+. Over
time, inactive acetylated MbtA regained its
activity when incubated with NAD+ and DAc.
However, omission of either NAD+ or deacetylase
prevented reactivation (Fig. 3B). These results
suggest that MbtA catalytic activity is regulated by
Pat and DAc via the reversible acetylation and
deacetylation of MbtA at position K546.
Crystal structure of Msmeg MbtA apo. Msmeg
MbtA was crystallized in an apo form and the
structure was solved to 2.3 Å (Table 1). The
Msmeg MbtA structure was solved by molecular
replacement using the DhbE structure (accession
code: 1MDB), which is the adenylation domain in
the bacillibactin siderophore biosynthesis (24).
Molecular replacement led to a partial solution for
MbtA with a defined N-terminal domain and two
C-terminal domain fragments (residues 450-462
and 478-503). To complete the Msmeg MbtA
structure, the C-terminal domain of DhbE was
aligned with the MbtA C-terminal fragments.
Through multiple cycles of refinements and
manual corrections, a Msmeg MbtA model was
generated containing one chain in the asymmetric
unit with residues 18-151, 157-313, 321-556
observed and with a Rwork of 18.9 % and a Rfree of
24.5 %. The overall structure of Msmeg MbtA
exhibits the typical fold observed for the ANL
(Acyl/Aryl, NonRibosomal Peptide Synthetases
and Luciferase) superfamily of adenylating
enzymes (25). The structure includes two main
domains (Fig. 5A): a large N-terminal domain (1-
457) and a smaller C-terminal domain (458-558).
The N-terminal domain is composed of three
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subdomains: two β-sheet subdomains (a and b)
and a β-barrel subdomain (c). The subdomains (a)
and (b) form a five-layered αβαβα sandwich. The
subdomain (a) includes six β-strands and five α-
helices while the second β-sheet subdomain
contains eight β-strands and seven α-helices. The
β-barrel subdomain (c) abuts (a) and (b) and leads
to the compact C-terminal domain through a short
hinge (including residue K457). The compact C-
terminal domain consists of three α-helices and
five β-strands and harbors K546, the lysine
acetylated by Pat.
The Msmeg MbtA structure is very similar to the
B. subtilis DhbE (44 % sequence identity) and the
Acinetobacter baumannii BasE N-terminal domain
(39 % sequence identity) structures.
Superimposition of MbtA with DhbE and BasE N-
terminal domain shows low r.m.s displacement
values for the Cα atom positions of 1.0 Å and 0.7
Å respectively (Fig. 5B) (24,26). The highly
conserved P-loop motif (S212-K221) is implicated
in the ATP phosphate moiety binding and adopts a
slightly different conformation compared to the
BasE N-terminal domain or the DhbE structures
due to the lack of substrate in the MbtA active site
(24,26). Salmonella enterica acetyl-CoA
synthetase (ACS) and MbtA both catalyzed
adenylation reactions and structural
superimposition indicates a fairly high structure
similarity (4.9 Å r.m.s displacement for the Cα
atom positions) (Fig. 5C) (27). Moreover, Pat
acetylates ACS-K609 and MbtA-K546, which are
both localized on the same C-terminal domain
loop. The main difference between the Msmeg
MbtA structure and the other structures (DhbE and
ACS) lies in the conformation adopted by the C-
terminal domain: the latter is rotated by 91° and
102° compared to the DhbE and the ACS C-
terminal domains, respectively. The C-terminal
domain forms a “lid”, which can close above the
β-barrel subdomain and plays a significant role in
the transfer of the adenylated product to the acyl-
carrier protein (24,28). This domain has been
shown to adopt different orientations depending on
which half-reaction is catalyzed, either the
adenylation or the thioesterification reactions
(25,29).
Generation of precise null deletion mutants in
genes encoding Pat (Rv0098) and DAc (Rv1151c)
in Mtb. Prior to this study, the potential for
regulation of mycobacterial siderophore
production via post-translational acetylation of
enzymes required for mycobactin biosynthesis has
been demonstrated biochemically. Here, we also
examine the in vivo impact of genetically
removing Pat or Dac (Fig. 6A and 6C), under
conditions encountered in the intraphagosomal
environment. Towards this end, using a BLS2-safe
M. tuberculosis H
37Rv derivative, which is a
double auxotroph for leucine and pantothenate, we
engineered ∆Pat and ∆DAc Mtb mutant strains
(Fig. 6B and 6D). The genes were deleted by the
specialized transduction methodology described in
the Materials and Methods. To verify the
deletions, PCR amplification using three
diagnostic independent primer pairs shows
expected amplicon sizes for ∆Pat + pMV261 and
∆DAc + pMV261 (Fig. 6E and 6F).
∆Pat and ∆DAc share distinct phenotypes at low
pH and limited iron conditions. Pat exhibits a
unique structural feature with an N-terminal
cAMP-binding domain fused to a C-terminal
GNAT domain. Our current study and past studies
(19,23) have shown that Pat acetylation activity is
dependent on cAMP generated by multiple
adenylate cyclases (AC). To investigate the effect
of Pat and DAc deletions in ∆Pat and ∆DAc, we
tested these strains under different physiological
conditions such as low pH at which specific ACs
induce cAMP production (22). As a reference
point, we first determined growth curves for the
five strains in Sauton medium at pH 7. We
observed no significant growth difference between
the Mtb WT + pMV261 (expression vector), Mtb
∆Pat + pMV261, Mtb ∆DAc + pMV261, Mtb
∆Pat + pMV261-Pat and Mtb ∆DAc + pMV261-
DAc (Fig. 7A). All strains carried the pMV261
expression vector in order to facilitate a
comparative analysis. In contrast, when these same
strains were grown in Sauton medium at pH 6,
Mtb ∆Pat + pMV261 showed a growth advantage
compared to the parent and complemented strains,
while Mtb ∆DAc + pMV261 displayed a slight
growth defect (Fig. 7B). These results indicate that
deletion of Pat leads to an acceleration of growth
and suggests that lack of acetylation of Pat
substrates at pH 6 is beneficial for Mtb. Since Pat
is able to potentially inhibit mycobactin
biosynthetic enzymes in vivo, we next cultured the
five strains in Sauton medium with limiting iron
(100 fold less than standard Sauton) to test the
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same hypothesis that inhibition of siderophore
production under these conditions would inhibit
growth in vivo. In iron-limited Sauton medium at
pH 7, we observed no significant difference in the
growth of the strains, but a much slower growth
rate was noted as compared with iron-sufficient
media (Fig 7C). If we now compared the growth
pattern in Sauton pH 6 with limited iron, we
observed a distinct growth advantage for Mtb ∆Pat
+ pMV261 compared to the parent and
complemented stains, while Mtb ∆DAc + pMV261
displayed a major growth defect (Fig. 7D). These
results support the idea that in vivo, at lower pH
and under elevated cAMP levels, that deletion of
Mtb Pat likely prevents the acetylation, and
inhibition of MbtA and FadD33, thus allowing for
mycobactin production, while deletion of Mtb
DAc likely prevents the deacetylation of these
enzymes and inhibits mycobactin production.
DISCUSSION
The acquisition of iron from the
environment, its incorporation into iron-containing
proteins and enzymes and the regulation of
intracellular iron levels is essential for the growth
and virulence of Mtb. While several methods for
iron acquisition exist in the organism, the major
mechanism is the production of the iron
siderophore, mycobactin. This secondary
metabolite has a number of unusual features. A
hydroxyphenyloxazoline moiety, made from
salicylate and serine, forms a N-cap to the adjacent
εN-hydroxylysine residue that is additionally
acylated on the ε-amino group. The remainder of
the molecule contains a hydroxybutyryl group and
a terminal εN-hydroxylysine residue that is
cyclized into a seven-membered hydroxamate
ring. The hexadentate coordination of ferric iron
occurs from the six oxygen and nitrogen atoms in
the molecule shown in blue in Figure 1A.
Acylation of the central hydroxylysine residue
occurs with saturated and Δ2-unsaturated long
chain fatty acids to generate mycobactin or with
short chain dicarboxylic acids to generate
carboxymycobactins. It is thought that the
carboxymycobactins are secreted and acquire host
iron and then deliver this iron to the membrane-
associated mycobactins (10).
It has been recognized for some time that
bacteria tightly regulate the production of
siderophores such as mycobactin. In Mtb, a major
regulator of mycobactin production is the iron-
binding transcriptional regulator IdeR (Rv2711).
This 230-residue protein is essential for
mycobacterial growth and is a member of the
diphtheria toxin repressor family and exists as a
homodimer in solution (30). It binds to a highly
conserved 19 base pair inverted repeat that is
found adjacent to genes whose transcription is
repressed at high iron concentrations. In both the
Mbt-1 and Mbt-2 gene clusters, multiple IdeR
binding sites are observed adjacent to the
transcriptional start sites for the genes in the two
clusters. As iron is acquired by the mycobactins
and the intracellular concentration increases, the
iron-bound IdeR binds to these regions, effectively
preventing transcription of these genes (31). This
is a general mechanism of regulation in many
bacteria. There is a recent report that the HupB
protein can serve as a positive regulator of
mycobactin synthesis under conditions of low
intracellular iron by binding to a region 5’ to that
of the IdeR binding site and recruiting RNA
polymerase to the transcription start site (32).
In the current study, we have analyzed the
potential for non-transcriptional regulation of
mycobactin biosynthesis. We have previously
reported the reversible, post-translational
monoacetylation of both the Mtb acetyl-CoA
synthetase (ACS) and the mbtM-encoded acyl-
ACP synthetase (FadD33) (19,23). These enzymes
are mechanistically related and use ATP to form
an intermediate acetyl or fatty acyl adenylate,
which is then nucleophilically attacked by the thiol
of CoASH or the phosphopantetheine arm of an
ACP domain. Knowing the primary sequence
around the lysine residue that is acetylated in both
these enzymes, we identified a highly similar
sequence in the Mtb and Msmeg MbtA enzymes. It
was previously shown that MbtA-K546 lysine
equivalents in FadD33 and ACS are required for
catalysis in the first adenylation half reaction
(19,23). The mechanism of MbtA is similar to that
of both ACS and FadD33 in that a carboxyl group
of the substrate is initially adenylated for
activation, followed by the attack of a thiol group
on the mixed carboxylic-phosphoric anhydride,
although MbtA uniquely uses an aryl carboxylate,
salicylate. We now show that MbtA is also
reversibly acetylated and that this leads to enzyme
activity loss.
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MbtA has become a well-established
druggable target in the last decade. The production
of mycobactin is essential and the tight binding of
hydrolytically stable, isosteric analogs of the
salicyladenylate has been demonstrated (33). The
intermediate analog, 5’-O[N-
(salicyl)sulfamoyl]adenosine was shown
independently by three groups to inhibit MbtA
with a Ki of ~ 6 nM (33-35). This compound
exhibits antimycobacterial activity in vitro with a
MIC value of 0.4 mM, and exhibited in vivo
activity in an acute mouse model. Since MbtA is
the first and committed step in mycobactin
synthesis, it is perhaps not surprising that its
inhibition would be so deleterious.
To support our biochemical analysis we
also performed genetic analysis to investigate the
role of acetylation in the Mtb physiological
contest. We generated two strains in which either
the Pat or DAc genes were knocked-out, and
strains in which the knockouts were
complemented. To mimic the environment inside
the macrophage phagosome, strains were grown at
low pH and limiting iron condition. While at pH 7
(Sauton pH7 and limited iron pH7), all strains
exhibit a similar growth phenotype. But at relevant
physiological pH (pH 6 and under limiting iron
conditions), the ΔPat strain grows significantly
faster than the WT, and the ΔDAc grows
considerably slower. Both complemented strains
grow approximately as well as WT. This suggests
that when acetylation of MbtA or FadD33 is
prevented in the ΔPat strain, the resulting active
enzymes are capable to generating the required
levels of mycobactin. On the other hand, in the
ΔDAc strain, MbtA and FadD33 are likely to be
functioning at less than full activity, even less
activity than the WT strain resulting in insufficient
mycobactin levels. Thus mutant phenotypes
suggest that acetylation may influence Mtb
survival in the macrophage.
It is clear that the transcriptional
regulation of the mycobactin biosynthetic gene
clusters is the major mechanism by which
mycobactin biosynthesis is regulated. However,
once the proteins are made, they remain
constitutively active, potentially enabling for the
production of excess mycobactin, and allowing for
the accumulation of intracellular iron in excess of
its ability to be incorporated into iron-dependent
proteins. This would lead to free iron in the cell,
and in the reducing, but aerobic, intracellular
environment of the cell, the potential for the
generation of reactive oxygen species through the
action of Fenton-type chemistry. In support of this,
a recent report showed that Mtb is particularly
sensitive to ascorbic acid, Vitamin C, in
comparison to other Gram negative and positive
species (36). The ability to rapidly inactivate
MbtA could be used to break the ATP-consuming
production of mycobactin very quickly when the
organism senses that sufficient iron is present.
This secondary level of regulation could be used to
fine tune iron levels, allowing just enough as
required and preventing the over accumulation of
iron to toxic levels.
EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of MbtA.
MbtA (MSMEG_4516 and Rv2384) was
amplified from Msmeg mc
2155 and Mtb H37Rv
genomic DNA using the primer pairs
Msmeg_MbtA_F (5’-
GGAATTCCATATGACTCTGACCACGCCCC
AC-3’) and Msmeg_MbtA_R (5’-
CCCAAGCTTCCCGCCGAGCTGACGCACGA-
3’) or Mtb_MbtA_F (5’-
GGAATTCCATATGCCACCGAAGGCGGCAG
AT-3’) and Mtb_MbtA_R (5’-
CCCAAGCTTATGGCAGCGCTGGGTCGTCA
C-3’) containing NdeI and HindIII sites,
respectively. The PCR amplicon was ligated into
the pET-28a (+) vector, and then transformed into
E. coli DH5α competent cells to create pET-28a
(+)::MbtA N-terminally His6 tag plasmid. A
sequence verified construct was transformed in E.
coli T7 express lysy/Iq for protein expression. A 4
mL preculture was used to inoculate 1 L of Luria-
Bertani medium supplemented with 50 µg/mL
kanamycin. The culture was grown to midlog
phase (A600 ~ 0.6) at 37 °C, then induced by the
addition of 0.4 mM isopropyl-1-thio-β-D-galacto-
pyranoside (IPTG). After 18 hrs of additional
incubation (20 °C), cells were harvested by
centrifugation (6,000 g, 20 min) and stored at -80
°C. The cell pellet was thawed and resuspended in
lysis buffer (25 mM Tris, pH 8.0 containing 150
mM NaCl, 10 mM imidazole) supplemented with
DNaseI (0.1 µg/mL), lysozyme (2 mg/ml) and
EDTA-free protease inhibitors cocktail (Roche).
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Resuspended cells were disrupted by sonication
and cellular debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. MbtA was
purified by nickel affinity chromatography. After
an extensive wash with 25 mM Tris, pH 8.0
containing 150 mM NaCl and 10 mM imidazole
buffer, the bound protein was eluted with a linear
imidazole gradient (30-250 mM). Pure fractions,
as determined by SDS-PAGE, were pooled
together for buffer-exchange into 25 mM Tris, pH
8.0 containing 150 mM sodium chloride and 10 %
glycerol. For crystallization purpose, the N-
terminal polyhistidine tag of Msmeg MbtA was
cleaved using thrombin protease. The cleavage
reaction was dialyzed overnight against 50 mM
Tris (pH 8.0), 150 mM sodium chloride, 1 mM
dithiothreitol and 1 mM calcium chloride. The
enzyme was further purified using size exclusion
chromatography (HiLoad
TM 26/60 SuperdexTM 75
prep grade) in the buffer described above without
calcium chloride and then concentrated to 12
mg/mL using a 30-kDa Amicon Ultra Centrifugal
filter.
Crystallization. The apo enzyme was screened
against the MCSG suite (Microlytic, 384
conditions). A Crystal Gryphon robot (Art
Robbins) was used to dispense both the reservoir
solutions and the sitting-drops (1:1 ratio). Crystal
trays (Intelli-Plates 96, Art Robbins) were then
sealed and incubated at 20 °C. Preliminary crystals
were obtained in the condition 1D2 containing 0.2
M sodium chloride, 0.1 M bis-tris (pH 6.5) and 25
% (w/v) polyethylene glycol 3350. The
crystallization condition containing 0.1 M bis-tris
(pH 6.5), 0.4 M sodium chloride and 17.5 % (w/v)
polyethylene glycol 3350 improved the quality of
the crystal. The latter was cryoprotected by
addition of 30 % (w/v) polyethylene glycol and
flash-cooled in liquid nitrogen.
Data collection and structure determination.
Diffraction data were collected at the Lilly
Research Laboratories Collaborative Access Team
(LRL-CAT) beamline at the Advance Photon
Source Argonne National Laboratory (APS-ANL,
IL), at 0.97931 Å wavelength radiation.
Diffraction was observed to 2.3 Å. Data was
indexed, integrated and finally scaled using the
XDS package (37). Matthews’ coefficient analysis
indicated the presence of one molecule in the
asymmetric unit (43 % solvent). The structure of
DhbE from B. subtilis (PDB entry 1MDF) was
used to carry out molecular replacement with
PHASER-MR (24,38). The solution obtained by
molecular replacement was used as a template for
the Autobuild tool to build 78 % of the model (39).
The rest of the model was manually built using
COOT and iterative cycles of refinements (rigid
body refinement, simulated annealing, positional
and B-factor refinements) (40,41). Three residues
were observed in the disallowed region of the
Ramachandran plot (V197, V544, R554).
Cloning, Expression, and Purification of MbtB-
ACP. The acyl carrier protein domain of MbtB
enzyme, MbtB-ACP (Rv2383c) was amplified
from Mtb genomic DNA using the primer pairs
Mtb_MbtB-ACP_F (5’-
GGAGGGCATATGGTGCATGCTACGGCG-3’)
and Mtb_MbtB-ACP_R (5’-
CCCAAGCTTTGCGGCAACTGCCGTGGG-3’)
containing NdeI and HindIII sites, respectively.
The PCR amplicon was ligated into the pET-28a
(+) vector, and then transformed into E. coli
DH5α competent cells to create pET-28a
(+)::MbtB-ACP N-terminally His6 tag plasmid. A
sequence verified construct was transformed in E.
coli T7 express lys
y/Iq for protein expression.
MbtB-ACP domain was expressed as per MbtA
and cells were stored at -80 °C. The cell pellet was
thawed and resuspended in lysis buffer (25 mM
Tris, pH 8.0) supplemented with DNaseI (0.1
µg/mL) and EDTA-free protease inhibitors
cocktail (Roche). Resuspended cells were
disrupted by homogenization using the
EmulsiFlex-C3 (Avestin) at 10000 psi and cellular
debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. MbtB-
ACP was purified by nickel affinity
chromatography. After extensive wash with 4 M
urea, 25 mM Tris, pH 8.0, 10 mM imidazole
buffer, the bound protein was eluted with a linear
imidazole gradient (30-250 mM) with 4M urea.
Pure fractions, as determined by SDS-PAGE were
pooled together for buffer-exchange first in 1 M
urea, 50 mM Tris, pH 8.0, 150 mM NaCl and then
in 50 mM Tris, pH 8.0, 150 mM NaCl to allow
refolding. MbtB-ACP was concentrated using an
Amicon pressure concentrator with a 3 K Dalton
filter to 100 µM.
Cloning, Expression, and Purification of Sfp. Sfp
was amplified from B. subtilis genomic DNA
using the primer pairs Bsub_Sfp_F (5’-
GGTATTGAGGGTCGCATGAAGATTTACGG
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AATT-3’) and Bsub_Sfp_R (5’-
AGAGGAGAGTTAGAGCCTTATAAAAGCGC
TTCGTA-3’). The PCR amplicon was ligated into
the pET-30 Xa/LIC (+) vector, and then
transformed into E. coli DH5α competent cells to
create pET-30 Xa (+)::Sfp N-terminally His6 tag
plasmid. A sequence verified construct was
transformed in E. coli T7 express lysy/Iq for protein
expression. A 4 mL preculture was used to
inoculate 1 L of Luria-Bertani medium
supplemented with 50 µg/mL kanamycin. The
culture was grown to midlog phase (A600 ~ 0.6) at
37 °C, then induced by the addition of 0.1 mM
IPTG. After 18 hrs of additional incubation (30
°C), cells were harvested by centrifugation (6,000
g, 20 min) and stored at -80 °C. The cell pellet was
thawed and resuspended in lysis buffer (50 mM
sodium phosphate, pH 8.0 containing 300 mM
NaCl, 10 mM imidazole) supplemented with
DNaseI (0.1 µg/mL), lysozyme (2 mg/ml) and
EDTA-free protease inhibitors cocktail (Roche).
Resuspended cells were disrupted by sonication
and cellular debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. Sfp was
purified by nickel affinity chromatography. After
an extensive wash with 50 mM sodium phosphate,
pH 8.0 containing 300 mM NaCl and 10 mM
imidazole buffer, the bound protein was eluted
with a linear imidazole gradient (30-250 mM).
Pure fractions, as determined by SDS-PAGE, were
pooled together for buffer-exchange into 50 mM
sodium phosphate, pH 8.0 containing 150 mM
NaCl, 5 mM DTT, 10 mM MgCl2 and 10 %
glycerol. Sfp was concentrated using an Amicon
pressure concentrator with a 10 K Dalton filter to
250 µM.
Phosphopantetheinylation of MbtB-ACP by Sfp.
To convert the apo form of MbtB-ACP to the
phosphopantetheinylated form, holo-MbtB-ACP,
the phosphopantetheinyl transferase Sfp was used.
100 µM Apo-MbtB-ACP was incubated with 0.2
µM Sfp in 50 mM Tris, pH 7.8 with 150 mM
NaCl, 1 mM CoASH and 2 mM DTT. The
reaction was allowed to proceed at 25 °C for 18
hrs before buffer-exchange into 50 mM Tris, pH
8.0, 150 mM NaCl. Traces of Sfp were removed
during MbtB-ACP concentration using two
successive Amicon filtrations, 30 K Dalton and 3
K Dalton cut-off filters respectively.
Measurement of Enzymatic Activity. The
enzymatic activity of MbtA was determined
spectrophotometrically by coupling the formation
of AMP to the reactions of myokinase, pyruvate
kinase and lactate dehydrogenase as described
previously (42). Reactions were performed in 100
mM Hepes pH 7.5, 10 mM MgCl2, 250 mM NaCl,
1 mM PEP, 0.15 mM NADH, 300 µM holo-MbtB-
ACP, 18 units of myokinase, 18 units of pyruvate
kinase, and 18 units of lactate dehydrogenase in a
final volume of 100 µL. Typically 0.5 µM of
Msmeg or Mtb MbtA was used and incubated for 5
min at 25 ºC with the reaction mix prior to
reaction initiation by substrate addition with 2,3-
dihydroxybenzoate, a salicylic acid analogue. The
reaction was monitored at 340 nm (ε340 = 6220 M-
1cm-1) using a Shimadzu spectrophotometer (UV-
2450).
Site-Directed Mutagenesis and Purification of
MbtA-K546A. The K546A mutation was
introduced into the pET-28a (+)::MbtA N-
terminally His6 tag plasmid using the Quickchange
mutagenesis kit (Stratagene) with the following
primers Mtb_MbtA_K542A_F (5’-
CAACGCCGATCGGGGCGATCGACAAACGA
G-3’), Mtb_MbtA_K542A_R (5’-
CTCGTTTGTCGATCGCCCCGATCGGCGTTG-
3’), Msmeg_MbtA_K546A_F (5’-
CACGGCCGTCGGCGCGATCGACAAGAAG-
3’) and Msmeg_MbtA_K546A_R (5’-
CTTCTTGTCGATCGCGCCGACGGCCGTG-
3’). The mutation was confirmed by DNA
sequencing. The expression and purification of
MbtA mutant was the same as described for the
wild type.
In vitro Acetylation Assay. 10 µM MbtA or the
mutant protein were incubated with 1 mM cAMP,
100 µM AcCoA and 1 µM Msmeg Pat at 37 °C for
5 hours. Samples were then analyzed by SDS-
PAGE. Proteins were transferred to a
nitrocellulose membrane. Western blots were
performed using an anti-acetyllysine antibody
(Cell Signaling Technology Inc. and
ImmuneChem Pharmaceuticals Inc., dilution of
1:2000) and Goat Anti-Rabbit IgG AP conjugate
(Bio-Rad Laboratories, dilution of 1:2000).
Development was carried out according to the
manufacturer’s instructions.
Time-Dependent Inactivation and reactivation of
MbtA. 20 µM MbtA was incubated with 1 mM
cAMP and 100 µM AcCoA with 5 µM Msmeg Pat
in 50 mM HEPES pH 7.5, 100 mM NaCl at 37 °C.
Aliquots of the reaction mixture were withdrawn
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every hour. The residual activity of MbtA was
measured as described above. Control experiments
were performed when Msmeg Pat or AcCoA were
not included. MbtA acetylation was also assessed
by western-blot. For reactivation time course, the
acetylated MbtA was then purified by Ni-NTA
chromatography and used in the deacetylation
assay. A typical reaction mixture contained 10 µM
acetylated MbtA, 2 mM NAD+, and 5 µM
Rv1151c (deacetylase). Aliquots of the reaction
mixture were withdrawn every hour and MbtA
activity was measured as described above. Control
experiments were performed without adding
NAD+ or the deacetylase. MbtA deacetylation was
also assessed by western-blot.
Mass Spectrometric Analysis of Acetylated MbtA.
The mass spectrometric analysis was performed at
the Laboratory for Macromolecular Analysis and
Proteomics (LMAP) of Albert Einstein College of
Medicine. The same protocol as described in
(19,43) was implemented for identification of
MbtA acetylation sites.
Media and Bacterial Cultures. Our growth studies
utilized a BSL2-safe derivative strain of Mtb
H37Rv called mc26206 (H37Rv ΔpanCD
ΔleuCD), which is routinely used by the Mtb
community (44). mc26206 is a strain auxotrophic
for both pantothenate and leucine (pan- leu
-) and
was maintained in Middlebrook 7H9 broth (Difco)
supplemented with 10 % ADC (5 % bovine serum
albumin [BSA], 2 % dextrose, 5 % catalase) 0.025
% tyloxapol, 50 µg/ml of pantothenate and 50
µg/ml of leucine. Antibiotics (hygromycin at 50
µg/ml and kanamycin at 25 µg/ml) were included
as appropriate for strains harboring antibiotic
resistance cassettes.
For growth in iron-limited medium,
mc26206 pan- leu
- strains were grown in
deferreted-Sauton. Sauton contains 0.2 % citric
acid, 0.05 % K2HPO4, 0.4 % L-asparagine, 0.2 %
dextrose, 6 % glycerol, 0.025 % tyloxapol, 50
µg/ml of pantothenate and 50 µg/ml of leucine. To
remove metal contamination, Sauton medium was
treated with Chelex-100 (Biorad) according to
manufacturer’s instructions. Chelex was removed
by filtration and the medium was supplemented
with 1 mg/L ZnSO4, 0.5 g/L MgSO4 and 16.2
mg/L ferric chloride. Iron limited Sauton medium
was supplemented with ZnSO4, MgSO4 as
described above and 0.162 mg/L of ferric chloride.
When required, kanamycin (25 µg/ml) and
hygromycin (50 µg/ml) were added to the
medium. Sauton, 7H9 and Sauton iron-limited
medium pHs were adjusted to 7.0 or 6.0 and 100
mM MES was added to maintain a constant pH
during growth curve.
Rv0998 and Rv1151c mutant constructions in Mtb.
The gene knockouts of Pat acetyltransferase
(Rv0998) and Sirtuin-like deacetylase DAc
(Rv1151c) were created by specialized
transduction methodology in mc26206 (44,45).
Constructs for allelic exchange were generated by
amplifying the upstream and downstream flanking
regions of each of the two genes using the primer
pairs listed in Table 2. The upstream and
downstream flanking regions were cloned into
suicidal delivery vector pYUB1471 to create an
allelic exchange vectors harboring a
selectable/counterselectable cassette [γδ(sacB-
Hyg)γδ] between the two flanking regions. The
allelic exchange constructs were incorporated into
shuttle mycobacteriophage vector phAE159; the
phasmid constructs used in this work were
obtained as part of a collaboration with Genomics
Institute of the Novartis Research Foundation
(GNF) to generate a set of gene deletion constructs
for Mtb (46). Phasmid DNA was electroporated
into Msmeg to obtain plaques at the permissive
temperature of 30 °C. Specialized transducing
phages were picked and amplified at 30 °C to
generate high titer mycobacteriophage. mc26206
was transduced with high-titer phages at the non-
permissive temperature of 37 °C to delete genes of
interest by specialized transduction (44,45). The
transductants were plated on selective medium:
Middlebrook 7H10 medium (Difco) containing
10% OADC enrichment (0.5 g oleic acid, 50 g
albumin, 20 g dextrose, 0.04 g catalase, 8.5 g
sodium chloride in 1 L water), 0.5% glycerol, 50
µg/ml leucine, 24 µg/ml pantothenate and 50
µg/ml hygromycin. Genomic DNA prepared from
transductants was screened by a three-primer PCR
to confirm gene deletion, using primers in Table 3.
For complementation, the Rv0998 and
Rv1151c genes were amplified by PCR from
H37Rv genomic DNA using the following primers
Rv00998_F
(TTTTTTTTGGATCCATTGGACGGGATAGC
CGAATTG), Rv0998_R
(TTTTTTTTGTTAACTCAGCCGACGGCCTCG
ATCAC), Rv1151c_F
(TTTTTTTTGGATCCAATGCGAGTGGCGGT
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GCTCAG), and Rv1151c_R
(TTTTTTTTGTTAACCTATTTCAGCAGGGCG
GGCAG) containing BamHI and HpaI
respectively. PCR products were digested with
BamHI and HpaI and cloned into the
mycobacterial episomal pMV261 vector using the
BamHI and HpaI restriction sites (47). After
sequence verification, complementation plasmids
were electroporated into the relevant deletion
mutant strains, with transformants selected on
7H10 medium as noted above, also containing 25
mg/ml kanamycin. To generate control strains for
comparison in growth studies, the pMV261 empty
vector was similarly electroporated into mc26206
parental, mc26206∆Pat and mc26206∆DAc.
Growth assays. Mycobacterial strains were
inoculated in liquid media, 7H9 pH 7 and then
transfered into fresh 7H9, Sauton or low-iron
Sauton media at either pH 7 or pH 6. After one
culture passage in the final medium to allow strain
adaptation, growth curves were started out at an
OD600 = 0.05 and optical densities were measured
at regular intervals for 20-40 days.
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AUTHOR CONTRIBUTIONS
OV, HX, JMT, JSB and WRJ planned and designed research. OV purified and LF crystallized MbtA
protein and determined its X-ray structure. OV and HX performed the in vitro enzyme activity. JMT
designed mutant constructs and mutants were obtained by HX and OV. OV, HX and AAM analyzed the
mutant phenotypes. OV, LF and JSB wrote the manuscript. All authors reviewed the results and approved
the final version of the manuscript.
The authors declare that they have no conflicts of interest with the contents of this article
ACKNOWLEDGEMENTS
We are grateful to Dr. Myrasol Callaway (Albert Einstein College of Medicine) for assistance in mass
spectrometry. We would like to thank Dr. Steven Almo and his laboratory for the use of their
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Mycobactin MbtA regulation!
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crystallography equipment.
FOOTNOTES
*This work was supported by NIH grants AI60899 (to J.S.B), AI26170, AI098925 and P01AI63537 (to
W.R.J).
**This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE)
Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory
under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access
Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly
Company, which operates the facility.
FIGURE LEGENDS
Figure 1. Salicyl-capped mycobactin siderophore structure, genetic loci and first biosynthesis
reaction. A, lipophilic mycobactin (MBT) and hydrophilic carboxymycobactin (cMBT) share a common
core structure but differ in the length of the alkyl substitution (R group). Atoms in blue are involved in the
hexadentate ferric iron coordination. B, mbt-1 gene cluster produces mycobactin core whereas mbt-2 gene
cluster assembles and loads acyl fatty acid onto mycobactin lysine core. The black boxes indicate the
presence of IdeR binding sequences, which cause genes repression upon iron binding on IdeR. C,
adenylation-ligation reaction catalyzed by MbtA. ACP : acyl carrier protein domain of MbtB.
Figure 2. Pat acetylates specifically MbtA on Lys-546. A, Partial alignments of MbtA proteins from
M.smegmatis (MSMEG_4516) and M. tuberculosis (Rv2384) versus M. smegmatis FadD33
(MSMEG_2132). Acetylated lysines are indicated in bold. Conserved residues and basic residues
flanking acetylated sites are underlined. B, wild type MbtA or MbtA K546A mutant was incubated with
multiple reaction components as indicated in the table. The samples were analyzed by western-blot
(bottom panel) with acetyl-lysine antibody (a-AcK) and total protein content was determined by Ponceau
red (top panel). C, MbtA unique acetylation site was identified by MS/MS as Lys-546. Shown in an
MS/MS spectrum charged tryptic peptide from Ms MbtA (TTAVGK
AcIDKK) bearing an acetylated
lysine. The acetylated lysine is indicated as KAc.
Figure 3. Acetylation inhibits and deacetylation enhances the MbtA enzyme activity. A, time-
dependent inactivation of Msmeg MbtA by acetylation. Msmeg MbtA activity was monitored at different
time intervals with the additional components: (●): cAMP, Msmeg Pat and acetyl-CoA; (▲): cAMP and
acetyl-CoA; (△): cAMP and Msmeg Pat. B, time-dependent reactivation of acetylated Mtb MbtA by
deacetylation. After acetylation by Msmeg Pat, Mtb MbtA was then incubated with the following
components: (●): NAD+ and Mtb DAc; (▲):Mtb DAc; (○): NAD+.
Figure 4. Mtb DAc (Rv1151c) enzyme deacetylates both MbtA homologues. A, acetylated Mtb MbtA
were analyzed by western-blot (bottom panel) with acetyl-lysine antibody (α-AcK) and total protein
content was determined by Ponceau red (top panel). B, acetylated Msmeg MbtA were analyzed by
western-blot (bottom panel) with acetyl-lysine antibody (α-AcK) and total protein content was determined
by Ponceau red (top panel).
Figure 5. Crystal structure of apo Msmeg MbtA. A, Stereo view of the overall structure of apo Msmeg
MbtA. The three subdomains parts of the N-terminal domain are colored in purple (a), blue (b) and cyan
(c), respectively, while the C-terminal domain, or “lid”, is represented in orange. The location of the
active site is highlighted by a star . The residue K546, demonstrated in this manuscript to be acetylated, is
rendered as sticks colored by CPK, with carbon atoms in orange. B, Superimposition of the apo Msmeg
MbtA (green), B. subtilis DhbE in complex with DHB adenylated (blue, pdb accession code: 1MDB) and
Acinetobacter baumannii BasE N-terminal domain (yellow, pdb accession code: 3O83) structures. The
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DHB-adenylate product is displayed as sticks colored by CPK, with carbon atoms in magenta. C,
Superimposition of the apo Msmeg MbtA (green) and Salmonella enterica acetyl-CoA synthetase in
complex with CoA and AMP (orange, pdb accession code 2P2F) structures. The CoA and AMP ligands
are rendered as sticks colored by CPK, with carbon atoms in white.
Figure 6. Construction and verification of Mtb Pat (Rv0998) and Mtb DAc (Rv1151c) deletion
mutants. A and C, Rv0998 and Rv1151c adjacent gene organisations are shown. B and C, the ORF
removal in ΔRv0998 and ΔRv1151c deletion strains are replaced by sacB-hygR cassette. E and F, left and
right flanking PCR were used to confirm deletion of Rv0998 and Rv1151c with specific set of primers.
Figure 7. Iron level and pH effects on Mtb Pat and Mtb DAc deletion mutant phenotypes. Four
growth conditions are presented: A, Sauton pH7 medium; B, Sauton pH6 medium; C, limited iron Sauton
pH7 medium and D, limited iron Sauton pH6 medium for five constructs. (black), Mtb wild type +
pMV261; (solid red), Mtb ∆Pat + pMV261; (dotted red), Mtb ∆DAc + pMV261; (solid green), Mtb ∆Pat
+ pMV261-Pat and (dotted green), Mtb ∆DAc + pMV261-DAc. Error bars represent standard deviations
from the mean of results from biological duplicates. Mtb wild type corresponds to mc26206.
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TABLES
Table 1. Data collection and refinement statistics.
Data Collection
MsMbtA apo
PDB ID
5KEI
Space Group
P212121
Unit Cell Dimensions
a, b, c (Å)
56.4; 85.8; 104.7
α, β, γ (°)
90.0; 90.0; 90.0
Resolution Range (Å)
50.0-2.3
Wavelength (Å)
0.97931
Rmerge (%) (Highest
shell)
12.8 (79.3)
CC(1/2) (Highest shell)
99.3 (65.5)
I/σI (Highest shell)
8.7 (1.6)
Completeness (%)
(Highest shell)
99.6 (98.8)
Multiplicity (Highest
shell)
3.9 (3.8)
Total Reflections
(Unique)
163230 (41974)
Refinement statistics
Rwork /Rfree
18.9/24.5
Number of non-
hydrogen atoms
Protein
3943
Water
102
Average B-factors (Å2)
40.3
Protein
40.3
Water
35.6
Wilson B factor (Å2)
35.8
R.m.s. deviations
Bond lengths (Å)
0.008
Bond angles (°)
1.169
Ramachandran plot
Favored (%)
95.0
Outliers (%)
0.6
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Table 2. PCR primers for cloning flanks of the indicated loci, to generate allelic exchange
substrates.
Locus
Flank
Primer
name
Primer sequence
Rv1151c
Left
DRv1151c
LL
TTTTTTTTCCATAAATTGGGCCGCGGACCTGGTAAATAA
Left
DRv1151c
LR
TTTTTTTTCCATTTCTTGGTACAAGAATTGCAGGCTGTG-
GATGTCTCACTGAGGTCTCTTCTTGTCATCGCGGAACGTC
Right
DRv1151c
RR
TTTTTTTTCCATCTTTTGGCCAAGATCCATGCCCTGACC
Right
DRv1151c
RL
TTTTTTTTCCATAGATTGGCTGGTCTAGTAGTGTATAGC-
CGAGTGTCTGGTCTCGTAGGACGATCAGCATCCGCGAGT
Rv0998
Left
DRv0998
LL
TTTTTTTTCCATAAATTGGACGAAGGCATTCCGGTCAAA
Left
DRv0998
LR
TTTTTTTTCCATTTCTTGGAATCGTATGACACGCGCTTGG-
ATGTCTCACTGAGGTCTCTCCGGACATCCCTGAAAGACG
Right
DRv0998
RR
TTTTTTTTCCATCTTTTGGAGCTGCGTCCCGGAAAAAGT
Right
DRv0998
RL
TTTTTTTTCCATAGATTGGTTAATGATTGTGGCTGACGCC-
GAGTGTCTGGTCTCGTAGGTCCGGGTGAGCTGAGCTTG
Table 3. PCR Primers used to screen for deletions at the indicated loci.
Locus
Flank
Primer name
Binding site
Primer sequence
Rv1151c
Left
1151c L Flank
F=P7
Upstream of L flank, in
wild-type and mutant
CAT GCC GTT CAG
CAT GTC
sacBout_LR=P10
Within AES construct,
mutant only
GAT GTC TCA CTG
AGG TCT CT
1151c L Flank
R= P8
Within deleted region,
wild-type only
GGT GAT GAC GCT
GAC CTC
Right
1151c R Flank
F=P5
Within deleted region,
wild-type only
CGT CAT CAC CCA
GAA TGT C
hygout_RR= P9
Within AES construct,
mutant only
CGA GTG TCT GGT
CTC GTA G
1151c R Flank
R=P6
Downstream of R flank,
in wild-type and mutant
GGC ACT GTC GGA
TTA CAA G
Rv0998
Left
0998 L Flank
F=P1
Upstream of L flank, in
wild-type and mutant
CGT TGT GTC TAC
TGC TCG AC
sacBout_LR=P9
Within AES construct,
mutant only
GAT GTC TCA CTG
AGG TCT CT
0998 L Flank
R=P2
Within deleted region,
wild-type only
GAT GAT CGC AAC
ACC ATC
Right
0998 R Flank
F=P3
Within deleted region,
wild-type only
CGG TTC ATG TCG
GCT CGT GTT C
hygout_RR=P10
Within AES construct,
mutant only
CGA GTG TCT GGT
CTC GTA G
0998 R Flank
R=P4
Downstream of R flank,
in wild-type and mutant
TGT GCG GTA CAT
CGA CCA CCT C
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R. Jacobs, Jr. and John S. Blanchard
Olivia Vergnolle, Hua Xu, JoAnn M. Tufariello, Lorenza Favrot, Adel A. Malek, William
Biosynthesis
Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore
published online August 26, 2016J. Biol. Chem.
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