Báo cáo khoa học: Loss of kinase activity in Mycobacterium tuberculosis multidomain protein Rv1364c pot

Loss of kinase activity in Mycobacterium tuberculosis
multidomain protein Rv1364c
Preeti Sachdeva
1,2
, Azeet Narayan
1
, Richa Misra
1
, Vani Brahmachari
2
and Yogendra Singh
1
1 Institute of Genomics and Integrative Biology (CSIR), Delhi, India
2 Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, India
Despite global efforts to control tuberculosis, it
remains an epidemic, with one-third of the world’s
population being infected by its etiologic agent, Myco-
bacterium tuberculosis, and over 1.5 million people
dying from the disease each year. The notorious suc-
cess of M. tuberculosis as a highly adapted pathogen
rests upon its ability to establish a persistent infection
in the hostile environment of the host cell through
mechanisms involving transcriptional reprogramming,
ensuring metabolic slowdown and upregulation of vir-
ulence and stress response pathways [1]. Switching of
alternative sigma factors is known to regulate global
gene expression to cope with the numerous environ-
mental conditions encountered during the establish-
ment of a successful infection [2]. The M. tuberculosis
genome encodes 13 sigma factors, including 10 alter-
Keywords

kinase; Mycobacterium; RsbW; Rv1364c;
SigF
Correspondence
Y. Singh, Institute of Genomics and
Integrative Biology, Mall Road, Delhi
110007, India
Fax: +11 2766 7471
Tel: +11 2766 6156
E-mail:
(Received 7 September 2008, revised 16
October 2008, accepted 22 October 2008)
doi:10.1111/j.1742-4658.2008.06753.x
The alternative sigma factors are regulated by a phosphorylation-mediated
signal transduction cascade involving anti-sigma factors and anti-anti-sigma
factors. The proteins regulating Mycobacterium tuberculosis sigma factor F
(SigF), anti-SigF and anti-anti-SigF have been identified, but the factors
catalyzing phosphorylation–dephosphorylation have not been well estab-
lished. We identified a distinct pathogenic species-specific multidomain pro-
tein, Rv1364c, in which the components of the entire signal transduction
cascade for SigF regulation appear to be encoded in a single polypeptide.
Sequence analysis of M. tuberculosis Rv1364c resulted in the prediction of
various domains, namely a phosphatase (RsbU) domain, an anti-SigF
(RsbW) domain, and an anti-anti-SigF (RsbV) domain. We report that the
RsbU domain of Rv1364c bears all the conserved features of the PP2C-
type serine ⁄ threonine phosphatase family, whereas its RsbW domain has
certain substitutions and deletions in regions important for ATP binding.
Another anti-SigF protein in M. tuberculosis, UsfX (Rv3287c), shows even
more unfavorable substitutions in the kinase domain. Biochemical assay
with the purified RsbW domain of Rv1364c and UsfX showed the loss of
ability of autophosphorylation and phosphotransfer to cognate anti-anti-

SigF proteins or artificial substrates. Both the Rv1364c RsbW domain and
UsfX protein display very weak binding with fluorescent ATP analogs,
despite showing functional interactions characteristic of anti-SigF proteins.
In view of conservation of specific interactions with cognate sigma and
anti-anti-sigma factor, the loss of kinase activity of Rv1364c and UsfX
appears to form a missing link in the phosphorylation-dependent interac-
tion involved in SigF regulation in Mycobacterium.
Abbreviations
GST, glutathione S-transferase; MBP, myelin basic protein; MursiF, multidomain regulator of sigma factor F; PAC, PAS domain-associated
C-terminus; PAS domain, Per-Arnt-Sim domain; pNP, p-nitrophenol; pNPP, p-nitrophenyl phosphate; PP, protein phosphatase; SigA,
sigma factor A; SigB, sigma factor B; SigF, sigma factor F; TNP-ATP, 2,4,6-trinitrophenyl ATP; UPD, RsbU ⁄ phosphatase domain; VSD,
RsbV ⁄ substrate domain; WKD, RsbW ⁄ kinase domain.
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6295
native sigma factors [3]. One of the alternative sigma
factors of M. tuberculosis, sigma factor F (SigF) is
responsible for transcription of gene products of
importance to infection and dormancy processes,
including genes involved in the biosynthesis of the cell
envelope and sigma factor C (SigC) [4]. An M. tuber-
culosis SigF-deleted strain grows to a three-fold higher
density in stationary phase than the wild-type strain,
and is attenuated for virulence in a mouse model of
infection [5].
The regulation of expression and activity of sigma
factors is brought about by phosphorylation and pro-
tein–protein interaction events in a partner-switching
mechanism involving anti-sigma factors and anti-anti-
sigma factors. M. tuberculosis SigF is related to sigma
factor B (SigB), a stress-response specific sigma factor
of Bacillus subtilis, and SigF of B. subtilis, a sporula-

tion-specific sigma factor [6]. In both the SigB and
SigF regulatory pathways of B. subtilis, the activity of
the sigma factor is negatively regulated by the cognate
anti-sigma factor, RsbW, and SpoIIAB, respectively,
which hold the transcription factor in an inactive com-
plex. Release of SigB and SigF from the complex is
mediated by the anti-anti-sigma factors, RsbV and
SpoIIAA, respectively. The action of the anti-anti-
sigma factors is counteracted by the kinase activity of
the dual-function RsbW and SpoIIAB proteins, which
phosphorylate and thereby inactivate their respective
anti-anti-sigma factors. Finally, the phosphorylated
RsbV and SpoIIAA proteins are reactivated by the
action of phosphatases, RsbU and SpoIIE respectively.
Other proteins, such as RsbT, act upstream by binding
and activating the phosphatase activity of RsbU in
B. subtilis [7–9]. Similarly, the M. tuberculosis genome
has been shown to possess a bona fide anti-SigF
protein, UsfX ⁄ RsbW, as well as two anti-anti-SigF
proteins, RsfA and RsfB. RsfB is regulated by phos-
phorylation, as a mutation that is believed to mimic
phosphorylation renders it nonfunctional. However,
RsfB is not phosphorylated by UsfX in an in vitro
kinase reaction [10]. The upstream molecules in this
event, i.e. a kinase and a phosphatase regulating
UsfX–RsfB interaction, remain to be elucidated.
The M. tuberculosis genome analysis reveals another
potential SigF regulatory gene, Rv1364c encoding a
protein with multidomain architecture, in which the
entire regulatory cascade akin to the RsbU–RsbW–

RsbV system in B. subtilis appears to be present within
a single polypeptide with an additional sensor domain,
the Per-Arnt-Sim (PAS) domain [11]. Rv1364c is
upregulated during nutrient starvation in M. tuber-
culosis [12], whereas its Mycobacterium bovis ortholog
is upregulated in response to environmental changes
encountered within the macrophages [11]. In a yeast
two-hybrid-based study, Rv1364c has been reported to
interact with SigF as well as UsfX, and interdomain
interactions between its RsbW and RsbV domains also
occur [13]. The role and mechanism of regulation of
the multidomain protein Rv1364c are intriguing, and
underline the need to study this component of the
M. tuberculosis SigF regulation network. The present
study focuses on the functional characterization and
role of Rv1364c in phosphorylation–dephosphorylation
of anti-anti-sigma factors, which are known to be
important for regulation of SigF in M. tuberculosis.
Results and Discussion
Domain and genomic organization of Rv1364c
orthologs
The gene product encoded by M. tuberculosis Rv1364c,
annotated as rsbU [3], represents a multidomain pro-
tein comprising fused units that occur as independent
stand-alone proteins in the same and other distant bac-
terial species. As the Rv1364c domain composition
represents a previously unexemplified unique fusion of
the sensor–RsbU–RsbV–RsbW module for SigF regu-
lation, we have renamed the protein as putative multi-
domain regulator of SigF (MursiF) (Fig. 1A). We

performed a comparative genomic analysis of
sequenced mycobacterial genomes to identify MursiF
orthologs across the Mycobacterium genus, using full-
length M. tuberculosis H
37
Rv MursiF as well as its
individual domains as query sequences. The analysis
revealed the conservation of MursiF orthologs across
all sequenced M. tuberculosis strains, namely M. tuber-
culosis H
37
Ra, M. tuberculosis F11, M. tuberculosis
CDC1551, M. tuberculosis C and all other sequenced
slow-growing pathogenic Mycobacterium spp., namely
M. bovis AF2122 ⁄ 97, Mycobacterium avium 104,
M. avium paratuberculosis K-10, Mycobacterium mari-
num and Mycobacterium ulcerans (Fig. 1A). MursiF
ortholog is, however, absent in Mycobacterium leprae,
where the gene encoding SigF itself is known to be a
pseudogene [14].
On the other hand, nonpathogenic fast-growing
Mycobacterium spp., such as Mycobacterium smegma-
tis, Mycobacterium gilvum, Mycobacterium vanbaalenii,
Mycobacterium sp. MCS, Mycobacterium sp. KMS,
and Mycobacterium sp. JLS, were found to have no
sequence homologous to the complete multidomain
module of MursiF. Search of the M. smegmatis gen-
ome using the RsbU domain of MursiF as a query
sequence revealed a protein annotated as response reg-
ulator receiver protein (MSMEG_6131). This protein

Functional characterization of Rv1364c P. Sachdeva et al.
6296 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS
comprises two domains, a phosphatase domain with
44% similarity to the MursiF RsbU domain, and a
receiver domain analogous to the phosphoacceptor
protein of histidine kinases (Fig. 1B). Unlike M. tuber-
culosis mursiF, the MSMEG_6131 gene possibly forms
an operon (intergenic distance 3 bp) with a gene
pair comprising a two-component system sensor
kinase (MSMEG_6130) and a response regulator
(MSMEG_6128) (Fig. 1B). Interestingly, adjacent and
in opposite orientation to this putative operon in
M. smegmatis, we identified two genes in tandem,
MSMEG_6129 and MSMEG_6127 encoding protein
sequences with 44 and 58% similarity to the RsbW
domain and the RsbV domain of M. tuberculosis
MursiF, respectively (Fig. 1B). The analysis of genomes
of other nonpathogenic Mycobacterium spp. namely
M. gilvum, M. vanbaalenii, Mycobacterium sp. MCS,
Mycobacterium sp. KMS, and Mycobacterium sp. JLS,
also revealed the conservation of sequences ortholo-
gous to the M. smegmatis response regulator receiver
protein (MSMEG_6131) and two-component system
(MSMEG_6130, MSMEG_6128) (Fig. 1B). The genes
encoding proteins homologous to the RsbW and RsbV
domains of MursiF (Fig. 1A) in these species were,
however, found to be located far apart from response
regulator receiver protein orthologs. The rsbW gene is
spaced precisely 25 genes away in the closely related
strains Mycobacterium sp. MCS, Mycobacterium sp.

KMS, and Mycobacterium sp. JLS, and 15 genes away
in the closely related species M. gilvum and M. vanba-
alenii, from the gene encoding the response regulator
receiver protein (Fig. 1B). These observations, very
interestingly, suggest that stand-alone RsbU, RsbW
and RsbV encoding genes in nonpathogenic mycobac-
terial species have converged to form a gene encoding
fused mutidomain RsbU–W–V module, specifically in
pathogenic Mycobacterium spp. The selective advan-
tage of domain fusion lies in the increased efficiency of
coupling and coregulation of the corresponding bio-
chemical reaction or signal transduction step as well as
expression of the fused domains [15]. It is likely that
radically fused genes, which may emerge as a result of
Membrane
protein
Rv1364c
RsfA
Mb1399
Membrane
protein
RsfA
MAV_1619
Acyl-CoA
dehydrogenase
Hypothetical
protein
MAP2361
Hypothetical
protein

Acyl-CoA
dehydrogenase
Acyl-CoA
dehydrogenase
PE-PGRS
family
pseudogene
MUL_3853
Acyl-CoA
dehydrogenase
PE family
protein
MM3991
M. bovis
M. tuberculosis
H
37
Rv
M. marinum
M. avium
paratuberculosis
M. ulcerans
M. avium
M. smegmatis
M. sp. KMS
M. gilvum
M. sp. MCS
M. sp. JLS
M. vanabaaleni
i

MSMEG_6131
Glucarate
dehydratase
Sensor
Kinase
Response
regulator
RsbW
RsbV
PAS domain
RsbU
/Phosphatase
d
omain (UPD)
RsbW/K
inase
d
omain (WKD)
RsbV/Substrate
d
omain (VSD)
Coiled coil
AB
domain
1
135
165
404
544
653

RsbU/Phosphatase domain
26
140
207
402
1
Hypothetical
protein
Response
regulator
Sensor
Kinase
Mmcs_2688
Hypothetical
protein
RsbW
RsbV
Response
regulator
Sensor
Kinase
Acyl-
transferase
Mflv_3268
Phospho-
diesterase
RsbW
RsbV
Response
regulator

Sensor
Kinase
Acyl-
transferase
Mvan_2987
Phospho-
diesterase
RsbW
RsbV
Hypothetical
protein
Response
regulator
Sensor
Kinase
Mjls_2718
Hypothetical
protein
RsbW
RsbV
Response
regulator
Sensor
Kinase
Hypothetical
protein
Mkms_2732
Hypothetical
protein
RsbW

RsbV
Signal receiver domain
Fig. 1. Schematic representation of domain architecture and genomic organization of Rv1364c and its orthologs in pathogenic Mycobacte-
rium spp. (A) and response regulator receiver protein orthologs in nonpathogenic Mycobacterium spp. (B). M. tuberculosis Rv1364c and its
orthologs are shown as (
) arrows, and M. smegmatis response regulator receiver protein and its orthologs are shown as ( )
arrows. The numbers below the domain architecture diagram (shown as boxes) refer to amino acids defining boundaries of each of the
domains. The two proteins share a common domain, RsbU ⁄ phosphatase domain (
). The sequences homologous to other two domains,
the RsbW domain (
) and the RsbV domain ( ), of Rv1364c (A) exist as independent genes in nonpathogenic Mycobacterium spp. (B).
represents the region containing a large number of genes separating the response regulator receiver protein and RsbW genes; 25
genes in Mycobacterium sp. MCS, Mycobacterium sp. KMS and Mycobacterium sp. JLS, and 15 genes in M. gilvum and M. vanbaalenii .
P. Sachdeva et al. Functional characterization of Rv1364c
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6297
genome rearrangements, fix in a population because
they have a novel function that is advantageous to the
organism [16]. Fusion of domains associated with a reg-
ulatory pathway for stress adaptation to form a contig-
uous polypeptide may be advantageous in the
evolutionary optimization of the genome of pathogenic
Mycobacterium spp. The unique pathogen-specific
domain architecture and its upregulation in Mycobacte-
rium residing in a macrophage environment [11] makes
MursiF a protein of utmost importance.
Sequence analysis of MursiF
In silico analysis was performed to determine the nat-
ure and domain features of M. tuberculosis H
37
Rv

MursiF. This 653 amino acid protein has an estimated
molecular mass of 69.523 Da and a pI value of 4.763.
MursiF is predicted to be a soluble protein by analysis
programs. MursiF has a PAS sensor domain at its
N-terminus, with an adjacent PAC (PAS domain-asso-
ciated C-terminus) region (Fig. 1A). PAS domains are
sensory modules that undergo conformational changes
in the presence of various physical stimuli and ligand
molecules [17]. PAS domains show conservation in
three-dimensional fold and dynamic properties rather
than in amino acid sequences [18]. The MursiF PAS
domain has 38% similarity to that of B. subtilis RsbP,
an energy stress-dependent serine phosphatase [19].
Interestingly, in addition to a PAS sensor domain and
RsbU–W–V module, MursiF has a coiled coil motif
spanning residues 135–165, as determined by the
multicoil [20] and smart [21] programs (Fig. 1A).
The coiled coil motif is a structural motif involved in
oligomerization [22], and may play an important role
in protein–protein interactions [23], which form the
fundamental basis of the SigF regulation mechanism.
The RsbU ⁄ phosphatase domain (UPD) of MursiF is
homologous to protein phosphatase PP2C-class ser-
ine ⁄ threonine phosphatase, and shows 43% similarity
with B. subtilis RsbU. Multiple sequence alignment of
MursiF and other similar bacterial PP2C family mem-
bers shows the conservation of all critical residues of
11 characteristic motifs, including those predicted to
be involved in divalent metal ion coordination, Asp211
(subdomain II) and Asp328 (subdomain VIII) (Fig. 2).

However, the sequence lacks the Va- and Vb-boxes of
the PP2C-type catalytic domain, as reported for the
RsbU, RsbX and SpoIIE family of phosphatases [24].
MursiF UPD possesses a PAS sensor domain at its
N-terminus (Fig. 1A) in place of a motif for interaction
with an activator, RsbT, that is present in Bacillus and
Staphylococcus homologs. During environmental stress,
RsbT positively regulates RsbU phosphatase activity
[25]. No sequences homologous to genes encoding the
RsbRST module are present in the M. tuberculosis
genome. In this scenario, recruitment of a signaling
domain, together with loss of a domain that mediates
stress-induced interaction with an activator, suggests a
direct sensing mechanism for stress signals by MursiF.
The putative anti-SigF domain of MursiF, the
RsbW ⁄ kinase domain (WKD), shows 40% similarity
to B. subtilis RsbW, a serine kinase belonging to the
GHKL family of kinases. This family of kinases is
defined by the presence of an ATP-binding fold called
the ‘Bergerat fold’, comprising three motifs, the
N-, G1- and G2-boxes, which have been found to be
conserved in histidine kinases and ATPases [26]. Anal-
ysis of the MursiF WKD sequence revealed conserva-
tion of most of the residues characteristic of the
N-, G1- and G2-boxes; however, some significant vari-
ations were observed. Careful comparative analysis of
MursiF WKD sequences across all sequenced Myco-
bacterium spp. and functionally characterized RsbW
proteins from other genera was therefore carried out
using multiple sequence alignment (Fig. 3A). We noted

that a region speculated to be a part of the ATP lid in
functional RsbW sequences of other genera is deleted
in the WKD sequences of all MursiF orthologs identi-
fied across Mycobacterium spp. (Fig. 3A). The ATP lid
changes its conformation on nucleotide binding, and is
presumed to couple ATP binding to function-specific
interdomain associations [27]. Mutagenesis of the pro-
posed hinge of the ATP lid in a GHKL family kinase,
EnvZ, has shown that this region is essential for kinase
activity [28]. Furthermore, a conserved lysine residue
close to the N-box of histidine kinases, which has been
shown to be important for nucleotide binding as well
as catalysis [29], is substituted in the WKD sequences
of all MursiF orthologs (Fig. 3A). The novel genes
formed as a result of fusion of two or more genes are
believed to experience a burst of rapid adaptive substi-
tution shortly after they are formed, followed by a
slowing of evolution, which is consistent with increased
evolutionary constraint [16]. Less than 50% similarity
of MursiF UPD and WKD and the aforementioned
divergence of MursiF WKD sequences from their
orthologous sequences in other bacteria may be attrib-
uted to adaptive evolution.
The M. tuberculosis genome encodes another func-
tional anti-SigF protein, UsfX (Rv3287c) [10], which
has insignificant similarity to the MursiF WKD
sequence. UsfX has been shown to catalyze phospho-
transfer to an artificial substrate, myelin basic protein
(MBP) [30], but not to any of the anti-anti-sigma fac-
tor proteins [10,30]. We carried out a comparative

analysis of UsfX sequences across all sequenced Myco-
Functional characterization of Rv1364c P. Sachdeva et al.
6298 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS
bacterium spp. and functionally characterized RsbW
proteins from other genera (Fig. 3B). We found that
M. tuberculosis UsfX, surprisingly, has substantially
divergent motifs (45% similarity to B. subtilis RsbW),
and completely lacks the G1-box consensus sequence
(Fig. 3B). Furthermore, the conserved amino acids in
the signature sequences of the N- and G2-boxes have
been substituted with other less similar residues
(Fig. 3B), and the possibility of functional competence
of these substitutions remains to be studied. Impor-
tantly, a Bordetella BtrW (RsbW ortholog) N-box
mutant has been reported to be incapable of phosphor-
ylating its substrate, BtrV (RsbV homolog), whereas
its BtrW G1-box mutant phosphorylated BtrV to a les-
ser extent than its wild-type counterpart and is also
defective in the ability to form a stable complex with
the phosphorylatable form of BtrV [31]. Similar to
MursiF WKD, the UsfX sequence also shows a dele-
tion of the ATP lid region as well as an absence of the
lysine residue close to the N-box of histidine kinases
(Fig. 3A,B). However, in view of the presence of sev-
eral divergent motifs in the UsfX sequence, it seems to
have acquired a large number of deleterious mutations
in an independent evolutionary event. In view of the
Fig. 2. Comparison of the MursiF RsbU ⁄ phosphatase domain with PP2C family serine ⁄ threonine phosphatases of similar classes from other
organisms. MursiF of M. tuberculosis was aligned with the PP2C domains of RsbU of B. subtilis, SpoIIE of B. subtilis and IcfG of Synecho-
cystis sp. using

T-COFFEE. Identical amino acids are indicated by asterisks, high similarity is indicated by double dots, and lower similarity is
indicated by single dots. The gaps are introduced to optimize the alignment and are indicated by the dashes. Various motifs described in the
text are marked and shown in bold. Two conserved aspartate residues, Asp211 and Asp328, involved in binding divalent cations and
mutated in the study are indicated as shaded residues.
P. Sachdeva et al. Functional characterization of Rv1364c
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6299
A
B
Fig. 3. Comparison of mycobacterial MursiF WKD sequences (A) and mycobacterial UsfX sequences (B) with functionally characterized
RsbW sequences from other genera. Alignments were done using
T-COFFEE. Identical amino acids are indicated by asterisks, high similarity is
indicated by double dots, and lower similarity is indicated by single dots. The gaps are introduced to optimize the alignment and are indicated
by the dashes. Various motifs described in the text are marked and shown in bold.
Functional characterization of Rv1364c P. Sachdeva et al.
6300 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS
aforementioned deletions and mutations, the ability of
MursiF WKD and UsfX protein to act as functional
kinases in the SigF regulation cascade is questionable
and needs to be addressed.
The putative anti-anti-SigF domain of MursiF, the
RsbV ⁄ substrate domain (VSD), has 50% similarity to
B. subtilis RsbV. The domain has been shown to inter-
act with MursiF WKD [13], and has all the conserved
features of the anti-anti-sigma factor sequence
(Fig. S1). A serine residue, Ser600, at the predicted
phosphorylation site is conserved in MursiF VSD
(Fig. S1).
Protein expression and purification
The above-mentioned in silico analysis necessitates bio-
chemical analysis of MursiF and UsfX. In order to

functionally characterize full-length MursiF as well as
each of its domains, the full-length M. tuberculosis
H
37
Rv mursiF gene and its individual domains, namely
upd, wkd, and vsd, were cloned and expressed as
His ⁄ glutathione S-transferase (GST)-tagged recombi-
nant proteins in Escherichia coli (Fig. 1A, Table 1). In
addition, His-tagged M. tuberculosis SigF and His-
tagged and GST-tagged M. tuberculosis UsfX were
also overexpressed in E. coli. All the proteins were
purified as hexa-His (H) or GST-fusion (G) proteins
(H-MursiF, H-UPD, H-WKD, G-WKD, H-VSD,
G-VSD, H-UsfX, G-UsfX, SigF) using affinity chro-
matography. MursiF and its phosphatase domain
(UPD) mutant proteins carrying aspartate residue
(D211A, D328A) mutations (H-MursiF-D211A,
H-UPD-D211A, H-MursiF-D328A, H-UPD-D328A)
were subsequently purified using the same strategy. On
electrophoretic analysis of the purified proteins,
H-MursiF (and its mutants), H-UPD (and its
mutants), H-WKD, G-WKD, H-VSD, G-VSD,
H-UsfX and G-UsfX were detected as 73, 47, 18, 44,
14, 37, 22 and 45 kDa proteins, respectively (Fig. 4).
These protein sizes were consistent with predicted mole-
cular masses along with appropriate hexa-His (3 kDa)
or GST (26 kDa) affinity tags, except for UsfX, which
migrated at a size slightly higher than the expected
molecular mass. The identity of purified proteins was
also confirmed by western blot using monoclonal anti-

bodies against penta-His and GST (data not shown).
MursiF UPD characterization
The phosphatase activity of purified full-length MursiF
(H-MursiF) and MursiF UPD was determined by its
ability to dephosphorylate a small molecule substrate,
p-nitrophenyl phosphate (pNPP), thereby forming a
p-nitrophenolate ion, which is detected by measuring
absorbance at 405 nm (Fig. 5A). Further characteriza-
tion revealed that MursiF phosphatase activity has a
pH optima of 8.5 and an optimum temperature of
37 °C (data not shown). The activity of H-MursiF was
found to be strictly Mn
2+
-dependent (Fig. 5B), being
maximal at 2.5 mm Mn
2+
(data not shown). Other
cations such as Ca
2+
,Ba
2+
,Zn
2+
,Sr
2+
,Co
2+
and
Ni
2+

failed to substitute for Mn
2+
, whereas Mg
2+
was found to inhibit pNPP hydrolysis by H-MursiF
(Fig. 5B). Two conserved aspartate residues, Asp211
and Asp328, at positions known to be involved in
metal ion coordination were mutated (Fig. 2). The two
Table 1. Summary of expression vectors used in the study.
Expression vector Description of protein expressed Name Amino acid Reference
pHTc-mursiF Full-length His-tagged MursiF H-MursiF – This study
pHTc-mursiFD211A H-MursiF carrying D211A mutation H-MursiF-D211A – This study
pHTc-mursiFD328A H-MursiF carrying D328A mutation H-MursiF-D328A – This study
pHTc-upd His-tagged MursiF RsbU ⁄ phosphatase domain H-UPD 1–404 This study
pHTc-updD211A H-UPD carrying D211A mutation H-UPD-D211A 1–404 This study
pHTc-updD328A H-UPD carrying D328A mutation H-UPD-D328A 1–404 This study
pHTc-wkd His-tagged MursiF RsbW ⁄ kinase domain H-WKD 404–544 This study
pGEX-wkd GST-tagged MursiF WKD G-WKD 404–544 This study
pHTc-vsd His-tagged MursiF RsbV ⁄ substrate domain H-VSD 545–653 This study
pGEX-vsd GST-tagged MursiF VSD G-VSD 545–653 This study
pHTc-usfX His-tagged UsfX H-UsfX – This study
pGEX-usfX GST-tagged UsfX G-UsfX – This study
pGEX-rsfB GST-tagged RsfB G-RsfB – This study
pGEX-Rv2638 GST-tagged Rv2638 G-Rv2638 – This study
pLCD1 His-tagged SigF H-SF – [6]
pHTc-sigA His-tagged SigA H-SA – This study
pGEX-pknB GST-tagged PknB G-PknB – [37]
P. Sachdeva et al. Functional characterization of Rv1364c
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6301
mutant proteins, H-MursiF-D211A and H-MursiF-

D328A, showed  10-fold lower activity than wild-
type MursiF (Fig. 6). This further emphasizes the
divalent cation dependence of MursiF activity and the
crucial role of Asp211 and Asp328 in Mn
2+
coordina-
tion. In an attempt to assign MursiF to a specific class
of phosphatases, the effect of various class-specific
phosphatase inhibitors, such as sodium orthovanadate,
calyculin A, cyclosporine, and okadaic acid, was stud-
ied. Insensitivity to okadaic acid, a potent inhibitor of
the PP2A and PP2B family of phosphatases, is one of
the unique characteristics of the PP2C subfamily [32].
None of the PP1, PP2A or PP2B class-specific inhibi-
tors, including okadaic acid, had any effect on the
phosphatase activity of MursiF. Only sodium fluoride,
a nonspecific phosphatase inhibitor, inhibited pNPP
hydrolysis by H-MursiF by 30% (Fig. 7). MursiF is
therefore an Mn
2+
-dependent PP2C-type alkaline
phosphatase that is most active in the physiological
temperature range. The purified phosphatase domain
(H-UPD) of MursiF was also studied, and was found
to have similar characteristics (data not shown).
P
ro
t
ei
n

m
ar
k
er
220
116
97
66
31
21
14.4
220
116
97
66
31
21
45
H-
M
u
rs i
F
H-
M
u
rs i
F
D2
1

1A
H-
M
u
rs i
F
D3 2
8A
H
-
UP
D
H-
WKD
G
-
WKD
G
-
V
S
D
H-
V
S
D
P
ro
t
ei

n
m
ar
k
er
H-
Us f
X
G
-
Us f
X
H-
S
F
Fig. 4. Analysis of recombinant proteins by
SDS ⁄ PAGE. Affinity-purified His-tagged (H-)
or GST-tagged (G-) fusion proteins were
subjected to 13% SDS ⁄ PAGE and stained
with Coomassie brilliant blue. The numbers
on the left indicate sizes of bands of molec-
ular mass marker. The proteins analyzed are
indicated at the top of each lane, and details
are listed in Table 1.
0
2
4
6
8
A

B
0 20 40 60 80 100 120
Time (min)
µM pNP formed/µg of
protein
0
0.2
0.4
0.6
0.8
1
1.2
1.4
No
ion
Mg Mn Ca Ba Ni Zn Co Sr
Divalent cation
(
5 mM
)

A
405
Fig. 5. Biochemical characterization of MursiF phosphatase activity.
(A) Time kinetics of phosphatase activity of MursiF were deter-
mined by the hydrolysis of a low molecular weight substrate,
pNPP, as measured by absorbance at 405 nm. The activity was
expressed as micromoles of pNP liberated ⁄ lg of protein. (B) Puri-
fied H-MursiF (5 lg) was incubated with pNPP in reaction buffer

containing different divalent cations (Mg
2+
,Ca
2+
,Ba
2+
,Zn
2+
,Sr
2+
,
Co
2+
, and Ni
2+
). A
405 nm
for each of the reactions at 100 min is plot-
ted. Each value is the average of three individual reactions and is
given as mean ± SD.
0
0.2
0.4
0.6
0.8
1
1.2
MursiF-
WT
MursiF-

D211A
MursiF-
D328A
Relative A
405
Fig. 6. Comparative analysis of phosphatase activities of wild-type
and mutant MursiF. MursiF-WT, unaltered ⁄ wild-type H-MursiF;
MursiFD211A, H-MursiF carrying the D211A mutation; Murs-
iFD328A, H-MursiF carrying the D328A mutation (Table 1). Con-
served aspartate residues (as marked in Fig. 2), predicted to be
involved in metal ion coordination, were mutated, and proteins
were assayed for pNPP hydrolysis. A
405 nm
for wild-type protein is
normalized as 1, and relative values for mutant proteins are plotted.
Each value is the average of three individual reactions and is given
as mean ± SD.
Functional characterization of Rv1364c P. Sachdeva et al.
6302 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS
To study protein dephosphorylation by MursiF,
[
32
P]serine ⁄ threonine-phosphorylated or [
32
P]tyrosine-
phosphorylated artificial substrates such as casein,
histone and MBP were incubated with H-MursiF and
H-UPD, but no dephosphorylation of any of the artifi-
cial substrates was seen. The reaction conditions were
varied over a large number of biophysical and bio-

chemical parameters. In all cases, the level of free inor-
ganic phosphate released from
32
P-phosphorylated
substrates on incubation with MursiF was found to be
insignificant and almost comparable to that seen in the
presence of H-MursiF-D211A or H-MursiF-D328A
(data not shown). In this regard, MursiF behaves dif-
ferently from other PP2C-type phosphatases, but its
refractory behavior to artificial substrates is in agree-
ment with that reported for the RsbU homolog in
Synechocystis sp. IcfG (Slr1860) [33].
Similarly, Bacillus RsbU and RsbX protein phos-
phatases display strict specificities for a single homolo-
gous phosphoprotein, RsbV and RsbS, respectively
[34]. Another member, SpoIIE, does not dephosphory-
late its physiological substrate protein, SpoIIAA,
following replacement of the phospho-serine residue at
its phosphorylation site by a phospho-threonine [35].
The lack of Va- and Vb-boxes in the PP2C-type cata-
lytic domain is a feature shared by MursiF UPD with
RsbU, RsbX and SpoIIE phosphatases, which are
known to be divergent PP2C-type phosphatases [24]
(Fig. 2). The residues in the Va- and Vb-boxes of the
PP2C phosphatase catalytic domain have not been
characterized for their role in catalysis, but their selec-
tive absence in RsbU-like phosphatases may possibly
be relevant for the stringent specificity of this group of
phosphatases.
MursiF WKD characterization

To examine whether MursiF retains kinase activity in
spite of the loss of critical residues involved in ATP
binding, as observed in our in silico analysis of MursiF
WKD (Fig. 3A), purified H-MursiF as well as the
H-WKD and G-WKD proteins were incubated with
[
32
P]ATP[cP] in appropriate buffer conditions. No
autophosphorylation signal was observed for any of
the proteins (data not shown). As expected, H-MursiF
or domain alone (H-WKD and G-WKD) did not
phosphorylate its purified RsbV ⁄ substrate domain (H-
VSD, G-VSD), despite the presence of the conserved
phosphorylation site Ser600 in VSD (Fig. S1). Also,
no phosphotransfer was seen on the artificial substrates
MBP and histone in the presence of H-MursiF or
H-WKD, despite attempts to standardize reaction con-
ditions and a longer exposure of the autoradiogram.
Purified M. tuberculosis UsfX also does not display
autophosphorylation or phosphotransfer to VSD.
Also, MursiF WKD and UsfX failed to phosphorylate
certain other anti-anti-SigF proteins, G-RsfB and
G-Rv2638 (Table 1) (data not shown). In the auto-
radiogram with both GST-tagged UsfX and WKD
preparations, an autophosphorylating kinase signal
was seen at a size lower than that of UsfX and WKD
(Fig. S2). This autophosphorylation signal and a low-
intensity signal for phosphotransfer on MBP seen in
Fig. S2 are not unique to the presence of UsfX or
WKD, as they are observed in the absence of these

proteins but with GST protein and other recombinant
nonkinase proteins, similarly purified from E. coli
(Fig. S2). Most notably, autophosphorylation as well
as phosphotransfer signals diminished after stringent
and extensive washing of resin-bound G-UsfX and
G-WKD with high salt (1 m NaCl) containing buffers
before protein elution (Fig. S2). The activity was there-
fore attributed to copurifying contaminating kinase(s)
from E. coli. Two independent groups have already
reported that M. tuberculosis UsfX, unexpectedly, is
impaired in its ability to phosphorylate its natural sub-
strates, anti-anti-sigma factors such as RsfA [10] and
Rv0516c [30].
MursiF WKD and UsfX were tested for their ability
to bind ATP by using a fluorescent ATP analog, 2,4,6-
trinitrophenyl ATP (TNP-ATP), which shows an
increase in fluorescence emission intensity accompanied
by a blue shift upon protein binding [36]. The fluores-
cence emission spectrum of TNP-ATP in the presence
of MursiF WKD and UsfX showed a small change in
0
1
2
3
No inhibitor
Sod.orthovanadate
Cyclosporine
Calyculin A
Sod. flouride
Okadaic acid

A
405
Fig. 7. Effect of various phosphatase inhibitors on MursiF phospha-
tase activity. Purified H-MursiF was preincubated with inhibitor
(sodium orthovanadate, 200 l
M; sodium fluoride, 50 mM; okadaic
acid, 100 l
M; cyclosporine, 5 mM; calyculin A, 1 lM) for 20 min at
25 °C in assay buffer prior to addition of pNPP. A
405 nm
for each of
the reactions at 100 min is plotted. Each value is the average of
three individual reactions and is given as mean ± SD.
P. Sachdeva et al. Functional characterization of Rv1364c
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6303
intensity as well as in wavelength of emission, whereas
the positive control, M. tuberculosis PknB, showed a
remarkable two-fold enhancement of the emission
intensity and a blue shift (from 556 to 545 nm)
(Fig. 8). M. tuberculosis MursiF WKD and UsfX
therefore show very weak ATP-binding ability. Given
this relatively low affinity of MursiF WKD and UsfX,
low intracellular ATP concentrations in normal physi-
ological conditions, and even lower ATP levels in the
hypoxic and nonreplicating state in mycobacteria [37],
their association with ATP in the cellular milieu for
kinase function is questionable.
To rule out loss of activity of purified MursiF WKD
and UsfX during purification or handling, and to check
for the presence of other features characteristic of anti-

sigma factors, we carried out interaction studies with
MursiF VSD and SigF using sandwich ELISA and an
Ni
2+
-nitrilotriacetic acid resin pull-down assay, respec-
tively (Fig. 9A,B). MursiF WKD was found to interact
with both MursiF VSD (Fig. 9A) and SigF (Fig. 9B),
in agreement with the results of Parida et al. [13]. The
interaction between MursiF WKD and SigF is specific,
as no interaction could be observed with the principal
M. tuberculosis sigma factor, sigma factor A (SigA)
(Fig. S3). Both MursiF WKD and UsfX show inter-
molecular interactions with themselves as well as with
each other (Fig. 9A). Similarly, UsfX interacts with
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
A

B
Blank
H-VSD
G-WKD

GST
G-WKD
G-UsfX
G-VSD
GST
G-UsfX
G-WKD
GST
H-VSD Blank H-VSD H-VSD H-WKD H-VSD H-UsfX H-UsfX H-UsfX H-UsfX H-UsfX
A
490
Added
Coated
H-SF bound – – – + + +
resin
G-UsfX G-WKD GST G-UsfX G-WKD GST
40% Input
Output
H-SF
Fig. 9. Interactions of MursiF domains with each other and with UsfX (A) and SigF (B). (A) In a sandwich ELISA-based assay, the GST-
tagged proteins (Table 1) were coated (Coated) on ELISA plates, and incubated with different concentrations of His-tagged interacting pro-
teins (Added). The bound proteins were probed with antibody against penta-His and developed using ortho-phenylenediamine. GST served
as negative control in the reactions. Each value is the average of three individual reactions and is given as mean ± SD. (B) Ni
2+
–Nitrilotriace-
tic acid resin-bound H-SF was incubated with purified GST, G-WKD and G-UsfX in separate reactions, washed extensively, and analyzed by
SDS ⁄ PAGE. Input lanes represent 40% of actual input protein added to H-SF-bound resin, and output lanes represent resin-bound proteins
obtained in the interaction assay.
0
500

000
1
000 000
1
500 000
2
000 000
2
500 000
450 500 550 600 650 700
Emission wavelength (nm)
Fluorescence intensity
1
2
3
4
Fig. 8. Fluorescence spectra of TNP-ATP in the presence and
absence of MursiF. Details of the experiment are given in Experi-
mental procedures. Curve 1: spectrum of TNP-ATP (8 l
M) in the
presence of buffer alone. Curve 2: TNP-ATP (8 l
M) in the presence
of H-WKD (1 l
M). Curve 3: TNP-ATP (8 lM) in the presence of
H-UsfX (1 l
M). Curve 4: TNP-ATP (8 lM) in the presence of PknB
(1 l
M).
Functional characterization of Rv1364c P. Sachdeva et al.
6304 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS

SigF (Fig. 9B); however, no interaction of UsfX with
MursiF VSD was detected (Fig. 9A). UsfX has been
shown to display selective interaction with anti-
anti-SigF proteins, as it interacts with RsfA and RsfB
[10], but not with Rv0516c [13]. Interaction of UsfX
with full-length MursiF, as observed earlier by Parida
et al. [13], can therefore be attributed to the interaction
between UsfX and the homologous domain of MursiF,
WKD, rather than the antagonist domain, VSD.
M. tuberculosis MursiF WKD and UsfX therefore
show most of the attributes of functional anti-sigma
factor proteins except for kinase activity.
In summary, we have demonstrated that the puta-
tive regulator of M. tuberculosis SigF, MursiF, has
retained phosphatase activity and its specific interac-
tion with SigF as well as the interaction between its
putative anti-sigma factor and anti-anti-sigma factor
domains but has lost its kinase activity. The absence
of critical residues and deletions in MursiF and UsfX
proteins across different species of Mycobacterium
could indicate that functional dissection of kinase and
anti-sigma factor activities is a phenomenon that is
conserved across members of the Mycobacterium
genus. As MursiF has phosphatase activity and, as
per our results, is likely to be very specific for its sub-
strates with regard to site and residue of phosphory-
lation, some other RsbW-like serine kinase may fill
this missing link in the phosphorylation-dependent
regulation of SigF by MursiF and UsfX in the Myco-
bacterium genus. Further work to identify protein

kinases that can compensate for this loss of activity
of mycobacterial anti-sigma factors is in progress.
Experimental procedures
In silico analysis
The completely sequenced genomes of the Mycobacterium
spp., available at NCBI ( />genomes/lproks.cgi) and the Sanger Centre (http://www.
sanger.ac.uk/Projects/Microbes/), were searched for the
presence of Rv1364c homologs by employing psi-blast [38]
and blastp programs ( />genom_table.cgi and />blast.shtml), with full-length M. tuberculosis H
37
Rv
Rv1364c protein and each of its independent domain
sequences as query sequences, using default parameters.
The domain structural organization of the retrieved
sequences was analyzed using smart (l
heidelberg.de/) [21], interproscan ( />InterProScan/) and conserved domain database (http://
www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Transmem-
brane segment analysis was performed using tmhmm
( and hmmtop
( Multiple sequence align-
ments were constructed using t-coffee [39].
Bacterial strains and growth conditions
M. tuberculosis H
37
Rv was cultured in Middlebrook 7H9
broth or 7H10 agar (Difco Laboratories, Detroit, MI, USA)
supplemented with oleic acid-albumin-dextrose-catalase,
Tween-80, and glycerol. E. coli DH5a and E. coli BL21 were
used for maintenance of plasmids and expression of recom-
binant proteins respectively. E. coli strains were cultured on

LB agar or broth with or without ampicillin.
Construction of expression plasmids and
mutagenesis
M. tuberculosis H
37
Rv genomic DNA was isolated and used
as template for amplification of the mursiF (Rv1364c) gene,
segments coding for MursiF individual domains (RsbU ⁄
phosphatase domain, upd; RsbW ⁄ kinase domain, wkd;
RsbV ⁄ substrate domain, vsd), usfX and rsfB and the
Rv2638 gene using flanking primers with appropriate
restriction sites (Table S1). The amplicons were digested
with restriction enzymes and cloned in pProEx-HTc (Invi-
trogen, Carlsbad, CA, USA) or pGEX-5X-3 (GE Health-
care Bio-Sciences, Uppsala, Sweden), previously digested
with the same enzymes. The plasmids used or constructed
in this study are listed in Table 1. The full-length mursiF
gene (pHTc–mursiF) and its phosphatase domain (pHTc–
upd) constructs were subjected to site-directed mutagenesis
using a QuikChange XL Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA, USA), in which two residues,
Asp211 and Asp328, were replaced by alanine using primers
carrying mutations (Table S1), and the resulting plasmids
were designated pHTc–mursiFD211A, pHTc–mursiFD328A,
pHTc–updD211A, and pHTc–updD328A (Table 1). The
plasmid coding for M. tuberculosis SigF, pLCD1 [6], was
kindly provided by W. R. Bishai (Johns Hopkins School of
Medicine, Baltimore, USA). The sequences of all clones
were confirmed by DNA sequencing.
Expression and purification of proteins

E. coli BL-21(DE3) cells were transformed with pLCD1,
E. coli BL-21 cells were transformed with all other con-
structs, and ampicillin-selected transformants were grown
at 37 °C under shaking, until the attenuance (D
600
)
reached 0.7 and induced with 1 mm isopropyl thio-b-d-
galactoside. All the proteins were purified as hexa-His (H)
and GST-fusion (G) proteins (H-MursiF, H-MursiF-
D211A, H-MursiF-D328A, H-UPD, H-UPD-D211A,
H-UPD-D328A, H-WKD, G-WKD, H-VSD, G-VSD,
H-UsfX, G-UsfX, H-SF, H-SA, G-RsfB, G-Rv2638, and
P. Sachdeva et al. Functional characterization of Rv1364c
FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS 6305
G-PknB) (as listed in Table 1), using Ni
2+
–nitrilotriacetic
acid resin and glutathione–Sepharose 4B resin, respectively,
according to the manufacturer’s instructions. The purified
proteins were analyzed by SDS ⁄ PAGE [40].
Phosphatase assay
The phosphatase activity of purified H-MursiF (5 lg) was
determined using hydrolysis of pNPP as described previ-
ously [41]. The effect of divalent cations (3 mm each) on
the activity of MursiF was examined by substituting Mn
2+
with other cations (Mg
2+
,Ca
2+

,Ba
2+
,Zn
2+
,Sr
2+
,Co
2+
,
and Ni
2+
) in the reaction. The optimal concentration of
Mn
2+
was determined by varying the concentration of
Mn
2+
(0–10 mm) in the reaction buffer. A standard curve
was plotted to calculate molar absorption coefficients, using
different concentrations of the reaction product, p-nitrophe-
nol (pNP). The phosphatase activity of H-MursiF on pNPP
was then calculated under optimal conditions in reaction
buffer (25 mm Tris, pH 8.5, 2 mm Mn
2+
,5mm dithiothrei-
tol) and plotted as number of micromol of pNP formed per
microgram of protein. The effect of class-specific phospha-
tase inhibitors on MursiF activity was examined. The vari-
ous inhibitors and their concentrations used were sodium
orthovanadate (2, 20 and 200 lm), sodium fluoride (5, 50

and 250 mm), okadaic acid (1, 10 and 100 lm), cyclospor-
ine (50 lm, 500 lm and 5 mm), and calyculin A (0.1 and
1 lm). Purified H-MursiF was preincubated with inhibitor
for 20 min at 25 °C in assay buffer prior to addition of
pNPP. The assay conditions were standardized with respect
to reaction pH (pH 6.0–9.0) and temperature (20–50 °C).
The protein phosphatase activities of H-MursiF, H-Murs-
iF-D211A and H-MursiF-D328A were determined by mea-
suring the release of P
i
from [
32
P]ATP[cP]-labeled artificial
substrates such as MBP, histone, and casein, as described
previously [41].
Kinase assay
The kinase activities of purified H-MursiF, MursiF
H-WKD, MursiF G-WKD, G-UsfX, H-UsfX and GST-
PknB (positive control) were determined using a modified
protocol as described previously [42]. The kinase reaction
contained 2 lg of each enzyme in the kinase buffer (50 mm
Tris ⁄ HCl, pH 7.6, 50 mm KCl, and 10 mm MgCl
2
, with
and without 10 mm MnCl
2
,1mm dithiothreitol, and
0.1 mm EDTA) with 10 lg of each substrate in the presence
of 5 lCi of [
32

P]ATP[cP] (BRIT, Hyderabad, India) and
20 lm unlabeled ATP in a total volume of 25 lL, and incu-
bated for 30 min at 25 °C. The reactions were stopped by
addition of SDS sample buffer, and proteins were separated
by 12% SDS ⁄ PAGE (autophosphorylation assay) or 15%
SDS ⁄ PAGE (substrate phosphorylation), and visualized by
autoradiography.
ATP binding
The ATP-binding properties of MursiF and RsbW were
studied using a fluorescent ATP analog, namely TNP-ATP
(Molecular Probes, Inc., Eugene, OR, USA) [36]. Experi-
ments were performed at 25 °C using a Jobin Yvon Fluoro-
Max-3 spectrofluorometer, with spectral bandwidths of 5
and 10 nm for excitation and emission, respectively.
H-MursiF, MursiF H-WKD, MursiF G-WKD, G-UsfX,
H-UsfX and GST-PknB (positive control) were suspended
in the above-mentioned kinase buffer at a concentration of
1 lm each, and equilibrated at 25 °C for 10 min. TNP-ATP
was added in increments of 1 lm to each of these proteins,
excitation was performed at 410 nm, and emission was
scanned in the 470–650 nm range. TNP-ATP binding was
determined as the increase in fluorescence at 555 nm in the
presence of protein. All spectra were corrected for buffer
fluorescence and for dilution (never exceeding 5% of the
original volume).
Sandwich ELISA
The GST-tagged proteins were coated on ELISA plate
(Maxisorb, Nunc, Naperville, IL, USA) in 50 mm carbon-
ate buffer overnight at 4 °C. The excess of protein was
washed out with NaCl ⁄ P

i
containing 0.05% Tween-20
(PBST), and wells were saturated with NaCl ⁄ P
i
containing
1% BSA (PBSB) for 2 h at room temperature. After six
washes with PBST, the coated proteins were incubated with
different concentrations of His-tagged interacting proteins
in the presence of binding buffer (50 mm Tris, pH 7.4,
200 mm NaCl, 0.02% NP-40, 10% glycerol). After six
washes with PBST, antibody against penta-His–horseradish
peroxidase monoclonal antibody (Qiagen, Hilden, Ger-
many) was added (1 : 7000 dilution in PBSB) and incubated
for 2 h in each of these wells. After six washes with PBST,
plates were developed as described previously [43].
Ni
2+
–Nitrilotriacetic acid resin-based interaction
of WKD with SigF
Purified SigF (H-SF) (7 lg) coupled to 100 lLofNi
2+
–
nitrilotriacetic acid resin (1 h at 4 °C with rotation) was
incubated with 7 lg each of GST-tagged WKD, GST-
tagged UsfX and GST protein (negative control) in binding
buffer (50 mm Tris, pH 7.4, 200 mm NaCl, 0.05% NP-40,
10% glycerol) (100 lL) at 4 °C for 30 min with constant
agitation. After washing with 1 mL of wash buffer (50 mm
Tris, pH 7.4, 300 mm NaCl, 0.1% NP-40, 10% glycerol)
five times, the resin containing bound proteins was resus-

pended in SDS sample buffer, boiled, separated on 12%
SDS ⁄ PAGE, and visualized by Coomassie staining. As a
control, G-WKD and G-UsfX were incubated with the
resin lacking H-SF under the same conditions to check for
Functional characterization of Rv1364c P. Sachdeva et al.
6306 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS
nonspecific binding of the protein to the Ni
2+
–nitrilotriacetic
acid.
GST pull-down assay for interaction of WKD with
SigA and SigF
Purified G-WKD and GST (7 lg each) was bound to
100 lL of GST resin (1 h at 4 °C with rotation) and incu-
bated with 7 lg each of H-SF and H-SA in binding buffer
(50 mm Tris, pH 7.4, 200 mm NaCl, 0.1% NP-40, 10%
glycerol) (100 lL) at 4 °C for 30 min with constant agita-
tion. After washing with 1 mL of wash buffer (50 mm Tris,
pH 7.4, 300 mm NaCl, 0.1% NP-40, 10% glycerol) five
times, the resin containing bound proteins was resuspended
in SDS sample buffer, boiled, separated by 12%
SDS ⁄ PAGE, and visualized by Coomassie staining.
Acknowledgements
Financial support was provided by Council of Scien-
tific and Industrial Research (NWP0038). P. Sachdeva,
A. Narayan and R. Misra are supported by a CSIR
fellowship. The pLCD1 plasmid was a kind gift from
W. R. Bishai, Johns Hopkins School of Medicine,
Baltimore, MD, USA.
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Supporting information
The following supplementary material is available:
Fig. S1. Comparison of mycobacterial MursiF RsbV ⁄

substrate domain (VSD) sequences with functionally
characterized RsbV sequences from other genera.
Fig. S2. Autoradiogram showing in vitro phosphoryla-
tion reactions using G-UsfX, G-WKD and GST (nega-
tive control) protein preparations.
Fig. S3. Specificity of interaction of MursiF WKD
with sigma factors.
Table S1. Summary of oligonucleotides used in the
study.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the
corresponding author for the article.
Functional characterization of Rv1364c P. Sachdeva et al.
6308 FEBS Journal 275 (2008) 6295–6308 ª 2008 Institute of Genomics and Integrative Biology. Journal compilation ª 2008 FEBS