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

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-

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

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.

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

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

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

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

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

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-

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-

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

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

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-

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

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

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

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

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 (http://www.ncbi.nlm.nih.gov/
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 (http://www.ncbi.nlm.nih.gov/sutils/
genom_table.cgi and http://www.sanger.ac.uk/DataSearch/
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 (http://smart.embl
heidelberg.de/) [21], interproscan (http://www.ebi.ac.uk/
InterProScan/) and conserved domain database (http://
www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Transmem-
brane segment analysis was performed using tmhmm
(http://www.cbs.dtu.dk/services/TMHMM/) and hmmtop
(http://www.enzim.hu/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)

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+

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

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,

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
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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