MoaB2, a newly identified transcription factor, binds to σ A in Mycobacterium smegmatis

Abstract

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.

IMPORTANCE
Mycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

Full text

| Genetics and Molecular Biology | Full-Length Text
MoaB2, a newly identied transcription factor, binds to σA in
Mycobacteriumsmegmatis
Barbora Brezovská,1 Subhash Narasimhan,2,3 Michaela Šiková,1 Hana Šanderová,1 Tomáš Kovaľ,4 Nabajyoti Borah,1 Mahmoud
Shoman,1 Debora Pospíšilová,1 Viola Vaňková Hausnerová,1,5 Dávid Tužinčin,2,3 Martin Černý,2,3 Jan Komárek,2,3 Martina
Janoušková,1 Milada Kambová,1 Petr Halada,6 Alena Křenková,7 Martin Hubálek,7 Mária Trundová,4 Jan Dohnálek,4 Jarmila
Hnilicová,1,5 Lukáš Žídek,3 Libor Krásný1
AUTHOR AFFILIATIONS See aliation list on p. 25.
ABSTRACT In mycobacteria, σA is the primary sigma factor. This essential protein binds
to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes.
Our knowledge about this factor in mycobacteria is limited. Here, we performed an
unbiased search for interacting partners of Mycobacterium smegmatis σA. The search
revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2
interaction was validated and characterized by several approaches, revealing that it
likely does not require RNAP and is specic, as alternative σ factors (e.g., closely
related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray
crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique,
unstructured N-terminal domain of σA was identied to play a role in the σA-MoaB2
interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but
not σB-dependent) transcription and may increase the stability of σA in the cell. We
propose that MoaB2, by sequestering σA, has a potential to modulate gene expression.
In summary, this study has uncovered a new binding partner of mycobacterial σA, paving
the way for future investigation of this phenomenon.
IMPORTANCE Mycobacteria cause serious human diseases such as tuberculosis and
leprosy. The mycobacterial transcription machinery is unique, containing transcription
factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small
RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription appara
tus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor,
and characterize its eects on transcription and σA stability. This information expands
our knowledge of interacting partners of subunits of mycobacterial RNAP, providing
opportunities for future development of antimycobacterial compounds.
KEYWORDS MoaB2, σA, mycobacteria, RNA polymerase, transcription
Mycobacteria are medically important Actinobacteria that contain human patho
gens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacte
rium abscessus. Mycobacterium (synonym Mycolicibacterium) smegmatis is a fast-growing,
non-pathogenic relative of these species (1, 2). Interestingly, it still contains a number of
genes recognized as important for virulence in M. tuberculosis (3). More importantly, M.
smegmatis and M. tuberculosis display high similarity in the composition of the transcrip
tion machinery, which mediates the rst step of gene expression, a process essential for
the functioning of the cell and its adaptation to changing environment.
In bacteria, the central enzyme of transcription is multisubunit RNA polymerase
(RNAP). The RNAP core consists of several subunits (α2ββ'ω) (4). This core is capable
of transcription elongation and termination but not initiation. To initiate, the RNAP
December 2024 Volume 206 Issue 12 10.1128/jb.00066-24 1
Editor Tina M. Henkin, The Ohio State University,
Columbus, Ohio, USA
Address correspondence to Jarmila Hnilicová,
jarmila.hnilicova@natur.cuni.cz, Lukáš Žídek,
lzidek@chemi.muni.cz, or Libor Krásný,
krasny@biomed.cas.cz.
Barbora Brezovská and Subhash Narasimhan
contributed equally to this article. Author order was
determined alphabetically.
The authors declare no conict of interest.
See the funding table on p. 26.
Received 22 February 2024
Accepted 18 September 2024
Published 5 November 2024
Copyright © 2024 Brezovská et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution 4.0 International
license.

core needs to bind a sigma factor (σ) to form the holoenzyme (5). This holoenzyme
recognizes specic DNA sequences called promoters from which transcription initiates.
Bacterial species typically contain various σ factors that provide specicity for dierent
promoter sequences. Prominent among these factors is the vegetative (primary) σ, σA
(σ70 in Escherichia coli) that is active mainly during the exponential phase of growth and
drives transcription of housekeeping genes. The other σ factors are usually referred to as
alternative σ factors.
Activities of alternative σ factors are regulated by anti-σ factors (6). They bind to σ
factors and block their binding to RNAP. Anti-σ factors consist of a σ-binding domain
and a signaling domain that responds to signals from the inside or outside of the cell.
They are poorly conserved at the sequence level and are often co-transcribed with
their respective σ factor genes (5). Primary σ factors typically do not have counterpart
anti-sigma factors, although the Rsd protein of E. coli was shown to bind to σ70 and
modulate gene expression (7).
M. smegmatis contains 28 σ factors (8, 9), most of which are still poorly characterized.
The primary σ factor, σA, consists of four domains (σAN, σA2, σA3, and σA4). Both M.
smegmatis and M. tuberculosis contain an alternative σ factor, σB, 64% identical with σA
(amino acid identity) that directs transcription of genes expressed in stationary phase
and during stress response and was also proposed to induce oligomerization of RNAP,
capturing it in an inactive conformation (10–14). Moreover, recently published work
characterizing M. smegmatis σB identied its involvement also during exponential phase
where σB binds to >200 promoter regions, including those driving expression of essential
housekeeping genes (15). σB, though, is not essential and diers from σA by the absence
of the N-terminal domain.
The E. coli σ70 N-terminal domain is called 1.1 and has a close-packed folded structure
(16). This structure is found in most bacterial species (17, 18). To the contrary, in
Actinobacteria including M. smegmatis, the N-terminal regions of primary σ factors
display divergent primary amino acid sequences and are predicted to be unfolded.
Hence, this domain in mycobacteria is not termed σA1.1 but σAN (19, 20). Functionally,
it was shown to be important for transcription in vitro and its absence negatively
aected survival (21). The exact mechanistic functioning and structure of this domain
in Actinobacteria are not yet dened.
Previous studies of mycobacteria have yielded discoveries of general factors required
for proper functioning of the transcription machinery, CarD and RbpA (22–29). These
proteins bind to RNAP and aect the stability of the transcription bubble during
initiation. Overall, however, our understanding of the mycobacterial transcription system
is still lagging behind that of the model organism E. coli. Here, as part of our eort to
extend our knowledge about mycobacterial transcription apparatus, we identify a new
interacting partner of M. smegmatis σA, MoaB2, determine its 3D structure, and show that
it does not bind to alternative σ factors but requires the N-terminal domain of σA for
the interaction. Subsequently, we demonstrate that while this protein is not essential, it
modulates mycobacterial transcription in vitro and may aect stability of σA in vivo.
RESULTS
σA binds/is in a complex with MoaB2
To start characterizing σA and the complexes it is present in, we used laboratory
wild-type (wt) strain M. smegmatis mc2 155 and fused the mysA gene (MSMEG_2758)
that encodes σA in its native locus with the sequence encoding the FLAG epitope. The
anity tag was positioned at the C-terminus of σA (σA-FLAG). Next, using the created
σA-FLAG strain and the parent wt strain without any FLAG (no FLAG strain as a negative
control), we pulled down proteins by immunoprecipitation (IP) from exponential and
stationary phases of growth with a monoclonal antibody recognizing the FLAG epitope.
The immunoprecipitated proteins were digested in solution and analyzed by liquid
chromatography coupled with mass spectrometry (LC-MS) that allows the characteriza
tion of complex protein mixtures with high sensitivity.
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Figure 1A and B shows enrichment (threshold log2 = 2) of proteins pulled down
from the strain containing σA-FLAG over proteins pulled down from the control strain
(non-specic binding). Pull-down from the exponential phase yielded a higher number
of signicantly enriched proteins than pull-down from stationary phase (Table S1).
Unsurprisingly, in both growth phases, σA was the most enriched protein as well as core
subunits of RNAP (α, β, and β’). RbpA, the transcription factor, was also highly enriched
in exponential phase. Other enriched proteins included, e.g., MoaB2 (MSMEG_5485), F420
(MSMEG_0777), CbiA (MSMEG_0067), and Pks (MSMEG_0408). These proteins have various
predicted functions that are not primarily associated with transcription (Table S1).
To identify the most prominent interacting partner(s), we repeated the IP experiments
and analyzed the immunoprecipitated proteins on SDS-PAGE (Fig. 1C). Consistent with
the previous results, a band of the size corresponding to MoaB2 was clearly visible in the
gels. The MoaB2 identity was veried by mass spectrometry.
MoaB2 was found to be highly enriched in both growth phases (Fig. 1A through C).
MoaB2 is a 17.9 kDa (pI 4.17) protein of unknown function. In E. coli, it has two homologs,
MoaB (30% aa identity) and MogA (29% aa identity). MoaB was originally believed to
be involved in the biosynthesis of molybdenum cofactor (Moco) (30) but subsequent
genetic and biochemical experiments revealed that it does not play any role in this
process (31, 32). MogA is involved in Moco biosynthesis (33).
To rule out the possibility that the σA-MoaB2 interaction was not an artifact caused by
the presence of the FLAG-tag on σA, we used a dierent antibody, this time recognizing
directly σA in the wt strain without any anity tag (no FLAG). Using stationary phase cells,
we observed that MoaB2 was found in complex with σA even in this genetic background
(Fig. 1D).
The mass spectrometry analysis also revealed that the MoaB2 protein interacting
with σA was 164 amino acids (aa) long and not 178 aa as annotated in Mycobrowser
(34) (MSMEG_5485) and Uniprot (A0R3I5). Based on our data, the translation of MoaB2
starts with a methionine (encoded by ATG) 14 aa downstream of the annotated putative
translation start (GTG; see Fig. S1).
We concluded that MoaB2 in the cell is shorter than the annotated version and binds
σA either directly or in complex with RNAP.
MoaB2 binds neither RNAP nor selected alternative σ factors
We next set out to determine whether MoaB2 binds to RNAP or interacts directly with
σA in the absence of RNAP and whether MoaB2 also binds alternative σ factors. For this
purpose, we used a M. smegmatis strain encoding a C-terminal FLAG-tag on the β subunit
of RNAP (strain LK1468) and created strains with ectopically integrated anhydrotetracy
cline (ATC) inducible genes for selected σ factors (FLAG-tagged) as well as the same type
of strain for σA. This approach was used to circumvent the need for specic conditions
associated with the activity of these σ factors. These factors were σB, σE, σF, σG, and σH.
σE (MSMEG_5072) together with σB (MSMEG_2752) were suggested to play roles in
transition to antibiotic-tolerant persistence and in situations when respiratory electron
transport chain is inhibited (35, 36). σF (MSMEG_1804), phylogenetically and functionally
similar to the general stress σB factor from Bacillus subtilis (37, 38), was implicated in
adaptation to stationary phase and conditions of heat and oxidative stress as well as
in carotenoid (isorenieratene) pigmentation associated with increased susceptibility to
hydrogen peroxide (39–41). The function of σG (MSMEG_0219) is still undened and σH
(MSMEG_1914) regulates a transcriptional network that responds to heat and oxidative
stress (42).
We performed IPs with anti-FLAG antibody from respective strains from exponential
and stationary phases and analyzed the results on SDS-PAGE (Fig. 2; Fig. S2). First, the
experiments revealed that MoaB2 was absent from a complex with RNAP that also
contained σA, strongly arguing that σA and MoaB2 do not interact in the context of
RNAP, at least not in detectable amounts. Second, the experiments showed that no other
tested alternative σ factor had pulled down MoaB2. An exception was σE in stationary
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phase where σE did not accumulate, likely being rapidly degraded. In this case, it was not
possible to make a conclusion.
σA and MoaB2 form a complex in vitro
Subsequently, we performed size exclusion chromatography (SEC) experiments to test
the ability of MoaB2 to form a complex with σA in vitro to reveal whether MoaB2 can
directly interact with σA, or whether another unknown factor is perhaps necessary for
the MoaB2-σA complex formation. Figure 3A and B show that σA eluted sooner than
MoaB2 when migrating alone. When mixed, a third peak appeared, migrating faster than
FIG 1 M. smegmatis MoaB2 is in the interactome of σA. (A) Volcano plots of proteins associating with M. smegmatis σA-FLAG (strain LK3207) pulled down in
exponential (EXP) and stationary (STA) phases of growth. The plots show LC-MS-identied proteins enriched in IP pull downs with anti-FLAG over proteins from
the control “no FLAG” strain (LK3016). Red spots indicate proteins signicantly enriched (−log10 P < 2, indicated with the horizontal dashed line; enrichment
>log2>2, indicated with the vertical dashed line). The spots show averages from three independent biological repeats. (B) Quantitation of relative enrichments
of selected σA-FLAG (LK3207) associating proteins from (A) compared to the “no FLAG” strain. Data from exponential (EXP) and stationary (STA) phases are
indicated. The bars show averages from three independent biological repeats. The SDs cannot be shown directly in the graph because they are calculated from
the intensity values, whereas the fold change is shown in the graph. However, the variance of the replicates is one of the parameters of the P-value calculated
in the t-test—the lower the variance, the lower the P-value (P-valueMoaB2_STA = 0.0036; P-valueMoaB2_EXP = 0.0037; P-valueα_STA < 0.001; P-valueα_EXP < 0.001;
P-valueβ_STA < 0.001; P-valueβ_EXP < 0.001; P-valueσA_STA = 0.0015; P-valueσA_EXP < 0.001). (C) SDS-PAGE of IPs of FLAG-tagged σA (LK3207) using the anti-FLAG
antibody. “No FLAG” strain is negative control (LK3016). Data from exponential (EXP) and stationary (STA) phases are shown. The dotted line indicates that
this panel was electronically assembled from two parts of one gel. MoaB2 is marked with red asterisk. The identity of the bands was determined by mass
spectrometry. The experiment was performed in three biological replicates [independent of experiments shown in (A)] with the same result. (D) SDS-PAGE of IP
of σA from the stationary phase “no FLAG” strain cells (wt, LK3016) using antibody against σ70 (anti-σ70, clone name 2G10). IgG is a mouse nonspecic IgG used
as a negative control. The band corresponding to MoaB2 is indicated with red asterisk. The identity of the bands was determined by mass spectrometry. The
experiment was performed in three biological replicates with the same result.
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σA (Fig. 3C), indicative of a larger complex. SDS-PAGE then conrmed the presence of
both proteins in the respective fraction (Fig. 3F). CarD, used as a negative control protein,
showed no interaction with MoaB2 (Fig. 3D, E and G). Thus, MoaB2 directly binds σA
without the need for an additional factor.
Stoichiometry of the σA:MoaB2 complex
Analytical ultracentrifugation was employed to determine the stoichiometry and
characteristics of the σA/MoaB2 interaction. Analysis of sedimentation velocity (SV) data
(Fig. S3A) revealed distinct size distributions for σA and MoaB2 proteins. σA was present
predominantly in the monomeric form (3.0 S peak), while the c(s) distribution of MoaB2
consisted of the major hexamer and a minor trimer peak (~4.8 S and 3.6 S). The frictional
ratio of σA (f/f0 ~ 1.55) was higher than the typical values for compact, globular proteins
and in agreement with the partially disordered σA structure. The addition of MoaB2 to
the σA sample led to a decrease in the σA monomer in solution and emergence of a new
(complex) peak. When σA was in molar excess, the s-value of the complex was ~10
S. At higher MoaB2 concentrations, the distribution shifted to lower sedimentation
coecients, revealing the formation of smaller complexes with incomplete saturation
by σA.
Stoichiometry of the interaction was determined from the dependence of the peak
area of the σA monomer on the increasing MoaB2:σA molar ratio. The analysis demon
strated a linear dependence of the peak area of free σA on the MoaB2:σA molar ratio
for values below 0.75, conrming an overall 1:1 (σA:MoaB2) binding stoichiometry (Fig.
S3B). This was consistent across experiments, underscoring high reproducibility. The
binding anity was relatively high, with a dissociation constant much lower than the σA
concentration used (~20 µM), in the submicromolar range (Fig. S3B).
To conrm these results, a multi-signal sedimentation velocity (MSSV) analysis (43)
of the molar ratio 1:0.3 of σA:MoaB2 was performed (Fig. S4). This procedure allowed
us to determine the relative stoichiometry of the interacting molecules in the complex
(provided that their spectral properties are suciently dierent). Here, we analyzed data
obtained at 280 nm (where only σA absorbed) and using interference optics (where
both proteins are detectable). The integration of the ~10 S complex peak after spectral
FIG 2 M. smegmatis MoaB2 does not associate with alternative σ factors. SDS-PAGE of IPs of FLAG-tagged sigma factors [σA (LK2073), σB (LK2077), σE (LK2157),
σF (LK2159), σH (LK2160), σG (LK2161), or FLAG-tagged β´ subunit of RNAP (LK1468)] using anti-FLAG antibody. The FLAG-tagged proteins were present in the
genome in an additional copy under ATC inducible promoter and expressed after ATC induction. In the case of σB (2×), twofold amount of cells were harvested
for IP to enhance the detection of MoaB2 if present. The “No FLAG” strain was used as a negative control (LK3016). Individual σ factors are marked with black
asterisks. Black arrows indicate respective anti-σ factors. MoaB2 is marked with red asterisk. The identity of the bands was determined by mass spectrometry.
Three independent experiments were performed with identical results. Visualization of proteins was done also by silver staining with the same result (Fig. S2).
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FIG 3 M. smegmatis σA interacts with M. smegmatis MoaB2 in vitro. All SEC runs were done using
Superdex 200 Increase 10/300 Gl column (GE Healthcare) calibrated using Blue dextran and six protein
standards ranging from 12.4 to 669 kDa selected from Gel Filtration Markers Kit for Protein Molecular
Weights (MW) 6,500–66,000 Da (Sigma-Aldrich, MWGF70) and Gel Filtration Markers Kit for Protein
Molecular Weights (MW) 29,000–700,000 Da (Sigma-Aldrich, MWGF1000). Void volume (V0) is marked.
(A) SEC chromatogram of σA from M. smegmatis. Peak elution volume was 12.6 mL, which suggests MW
of ~122 kDa. Fractions eluted at 12 to 13.5 mL were pooled and used for formation of the σA-MoaB2
complex. (B) SEC chromatogram of MoaB2 from M. smegmatis. Peak elution volume was 13.6 mL, which
suggests MW of roughly 77 kDa. Fractions eluted at 13 to 14.5 mL were pooled and used for formation
of the σA-MoaB2 complex. (C) SEC chromatogram of σA-MoaB2 complex formed by mixing samples from
(A) and (B). σA and MoaB2 were mixed at a molar ratio (monomer:monomer) of 1:2. Peak elution volume
corresponding to σA-MoaB2 complex was 10.4 mL, to unbound σA it was estimated to be 12.4 mL and
to unbound MoaB2 13.6 mL. These elution volumes indicate MW of approximately 330 kDa,133 kDa,
and 77 kDa, respectively. σA elutes before MoaB2 at a lower volume than expected according to its MW
because it is a non-globular protein with long loops (the longest intramolecular distance in the structured
part of σA is ~116 Å) and contains an intrinsically disordered N-terminal domain. The hexamer of MoaB2
is globular (the longest intramolecular distance ~80 Å). Individual peaks are marked with respective
proteins, and corresponding fractions used for SDS-PAGE analysis (see Panel F) are indicated with red
numbers. (D). SEC chromatogram of CarD from M. smegmatis. Peak elution volume was 16.3 mL that
suggests MW of ~23 kDa. Fractions eluted at 16–16.5 mL were pooled and used in E to test whether
MoaB2, under the used experimental conditions, does not form unphysiological complexes. The fraction
used for SDS-PAGE analysis (see Panel G) is indicated with the red number. (E) SEC chromatogram of
CarD and MoaB2 from M. smegmatis. Molar ratio of CarD and MoaB2 was approximately 1:1 (see Panel G).
Complex between MoaB2 and CarD is not forming as there was no new peak present, and the amount of
CarD eluting at the position of “free” CarD (16.3 mL) in both experiments is the same. Peak elution volume
corresponding to MoaB2 was 13.5 mL which suggests MW of ~81 kDa. Individual peaks are marked with
respective proteins, and corresponding fractions used for SDS-PAGE analysis (see Panel F) are indicated
with red numbers. (F) SDS-PAGE analysis of the σA-MoaB2 complex formation. Lines 1–3 contain selected
fractions from (C). MoaB2 in line 2 is present due to the tailing of σA-MoaB2 complex
(Continued on next page)
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decomposition gave an almost identical concentration of both components (5.62 µM σA,
4.45 µM MoaB2), supporting the overall 1:1 stoichiometry.
Determination of crystal structure of M. smegmatis MoaB2
To characterize M. smegmatis MoaB2 structurally, we cloned, overexpressed, and puried
MoaB2. MoaB2 crystals were screened for diraction, and diraction data were collected
(Table S2). The structure of M. smegmatis MoaB2 was solved by molecular replacement
using Mycobacterium marinum MoaB2 (PDB id: 3rfq; sequence identity of 86% with M.
smegmatis MoaB2) as a search model (44) (Fig. 4A and B). In the crystal, MoaB2 was
present as a hexamer. The nal crystallographic model was rened to an Rwork of 0.192
(Rfree 0.260). The data collection and renement statistics from the nal coordinates are
summarized in Supplementary Table (Table S2). Each subunit of the hexamer exhibited a
weak or missing electron density for the N-terminal residues 1–7, the C-terminal His-tag,
and linker residues from the plasmid (165–172). Therefore, the mentioned residues could
not be built properly.
The structure of each subunit of M. smegmatis MoaB2 is composed of a central
β-sheet that is slightly curved and is surrounded by six α-helices and one 310 helix. The
electron density of chains A and F is poor, and thus, the 310 helix is not visible. The
β-sheet is made up of ve parallel strands (β1–β4 and β6) and one antiparallel strand
(β5), which are located between the inner β4 strand and the adjacent β6 strand (Fig. S5).
The overall architecture of the protein subunit is similar to other known molybdopterin
(MPT)-adenylyl-transferase enzymes.
The hexamers are made up of two trimers (chains ABC and DEF) making contacts
through mostly hydrophobic and salt-bridge interactions at the interface between the
subunits. Assembly of subunits into trimers within the hexamer is strengthened through
several hydrogen bonds between Arg95 and Arg120 of one monomer and Val79, Thr80,
and Pro81 of another monomer (Fig. 4C). Interactions between the two trimers (trimer
ABC and trimer DEF) within the hexamer are mainly mediated by residues of α4- and
α7-helices and the turn residues following α4-helix. Residues Leu31, Glu34, and Glu38 in
α4-helix and Arg138 and Arg142 in α7-helix form the central core of the trimer-trimer
interface (Fig. 4D). The residues forming the trimer-trimer interface are not conserved in
all bacteria. Interestingly, the bacterial homolog MogA exists as a trimer both in crystal
and solution. The eukaryotic orthologs, G-domain of Cnx1 from plants (Cnx1G), and
G-domain of the multifunctional mammalian protein gephyrin were also shown to be
trimers (45).
In crystal, the M. smegmatis MoaB2 hexamer forms the asymmetric unit. This diers
from other MoaB structures (including its closest M. marinum homolog used as the
search model in this study) with dimers or trimers in the asymmetric unit and forming
hexamers through crystallographic symmetry. Nevertheless, all bacterial MoaB proteins
are hexamers in solution (30, 46). SEC (Fig. 3B) and dynamic light-scattering experiments
(Fig. S6) showed that M. smegmatis MoaB2 is a hexamer in solution as well. Calcula
tions using the Adaptive Poisson-Boltzmann Solver (47) showed that the electrostatic
potential on the surface of M. smegmatis MoaB2 is prominently negative (Fig. S7) (32, 48).
Structural similarity search using the DALI server (49) performed on MoaB2 then
revealed a high level of homology with several proteins involved in Moco synthesis. The
highest level of structural homology was observed with MoaB from M. marinum (PDB:
3rfq, Z-score of 32.9; rmsd of 0.4 Å over 153 Cα atoms). Among other homologs than
Fig 3 (Continued)
peak which therefore overlaps with the peak of free σA at the elution volume from which the fraction was
taken for the SDS-PAGE analysis. Color Prestained Protein Standard, Broad Range (New England Biolabs)
was used as a marker. The experiment was performed in three independent replicas with identical results.
(G) SDS-PAGE analysis of the MoaB2-CarD. Lines 4–6 contain selected fractions from (D)and (E). SDS-PAGE
was performed under same conditions as in Panel F.
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MoaB, Molybdenum cofactor biosynthesis protein (MogA) from Mycobacterium ulcerans
(PDB: 4twg, Z-score of 26.7; rmsd of 1.3 Å over 149 Cα atoms), Cnx1G (PDB: 1uux, Z-score
of 26.1; rmsd of 1.3 Å over 152 Cα atoms from Arabidopsis thaliana) (50), G domain of
gephyrin (PDB:1jlj, Z-score of 25.4; rmsd of 1.4 Å over 152 Cα atoms from Homo sapiens)
(45), and the largest domain of MoeA (PDB: 1g8l, Z-score of 10.9; rmsd of 2.9 Å over 133
Cα atoms from E. coli) (51) showed closest similarities.
σAN is important for the σA-MoaB2 complex: interaction in the cell
A major dierence between σA and σB (as well as the other σ factors) is the absence of the
N-terminal domain found in σA (σAN) from σB (52) (Fig. 5A). Because we did not detect any
binding of MoaB2 to σB (Fig. 2), we speculated that σAN may play a role in the interaction
with MoaB2. Moreover, the negative charge of the surface of mycobacterial MoaB2 as
revealed by the 3D structure suggested its potential interaction with the charged σAN,
especially with the 60 N-terminal positively charged aa.
To test the importance of the N-terminal domain of σA for the interaction with MoaB2,
we created two strains with ectopically integrated ATC inducible variants of σA lacking
σAN (σAΔN-FLAG) or σAN lacking the 60 N-terminal aa (σAΔ60aaN-FLAG). IP experiments
with these and control strains analyzed on SDS-PAGE showed that in exponential phase,
only full-length σA was able to pull-down MoaB2 (Fig. 5B). In stationary phase, both
full-length σA and σAΔ60aaN pulled down MoaB2, whereas σAΔN was unable to do so (Fig.
FIG 4 3D structure of M. smegmatis MoaB2. (A) Top view of the MoaB2 hexamer with the subunits shown in dierent colors. (B) Side view of the MoaB2 hexamer
colored as in Panel A. (C) Detail of the interactions between monomeric subunits in the MoaB2 trimer. The hydrogen bond contacts at the interface (residues
Val79, Thr80, and Pro81 from chain C and Arg95 from chain A) are shown, and the distances are specied in Å. (D) Detail of the trimer-trimer interface of MoaB2.
The hydrogen bond and salt bridge contacts at the trimeric interface between Glu34, Glu38, Arg138, and Arg142 from chain E and chain C are shown.
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5B). The relative amount of MoaB2 pulled down with σA was higher in stationary than in
exponential phase (Fig. 5C).
To verify the absence of the interaction between σAΔN and MoaB2 in stationary phase,
we performed another set of pull-down experiments with strains containing full-length
σA and σAΔN (Table S3). The pulled-down proteins were digested in solution and analyzed
by LC-MS. Figure 5D shows high and statistically signicant enrichment of MoaB2 with
full-length σA but not σAΔN.
Collectively, these experiments suggested that σAN is involved in the interaction
between σA and MoaB2. Moreover, the 60 N-terminal aa do not seem to be essential for
this interaction. Rather, the 61–160 aa region likely plays a role.
σAN is important for the σA-MoaB2 complex: NMR analysis
As mycobacterial σAN had been predicted to be unstructured (19) (Fig. S8), we deci
ded to test this prediction experimentally with puried protein samples in vitro. First,
the disordered nature of σAN was conrmed by circular dichroism (CD) measurement,
yielding a spectrum typical for random coil conformation, substantially dierent from a
spectrum of the mostly helical domain 1.1 of σA from B. subtilis (Fig. 6A). Next, the 1H-15N
HSQC NMR spectrum of σAN (residues 1–160) was acquired. The low dispersion of proton
chemical shifts of backbone amides in the recorded spectrum (Table S4) conrmed that
σAN adopts a disordered conformation (black contours in Fig. 6B).
To explore the importance of σAN for the σA-MoaB2 interaction, we recorded 1H-15N
HSQC NMR spectra (53, 54) of σAN (residues 1–160) alone and of σAN (residues 1–160)
in the presence of MoaB2 (twofold molar excess). No signicant dierence between the
spectra was observed (data not shown). The fact that the chemical shifts of σAN were not
aected by the presence of MoaB2 indicates that σAN alone does not form a suciently
strong complex with MoaB2.
Therefore, as the next step, 1H-15N HSQC NMR spectra of free full-length σA and of
full-length σA in the presence of MoaB2 were recorded (blue contours in Fig. 6B). Most
peaks in the spectrum of free σA corresponded to peaks of isolated σAN (consisting
of amino acids 1–160), indicating that the majority of σA forms a folded structure of
a size undetected by the 1H-15N HSQC experiment. Among several additional peaks,
six corresponded to three amides of three side-chains of Asn or Gln outside σAN, with
chemical shifts of side-chain amides in well-ordered proteins. The peaks of the σAN were
sharp as expected for disordered residues, with the exception of approximately 30 highly
conserved residues of the C-terminal region of the σAN.
As σA was not stable in solution at higher concentrations, the sample containing only
the σAN was used to assign the σA peaks. Combining 5D HabCabCONH, triple resonance,
and 15N-edited TOCSY and NOESY experiments, we obtained the assignment of 42%
non-proline residues.
The addition of MoaB2 to σA changed several features of the spectrum: (i) peaks of
side-chain amides outside the σAN disappeared (Fig. 6C), indicating the formation of
a larger rigid structure; (ii) specic shifts of peak positions were observed (labeled by
arrows in Fig. 6D and E, summarized in Fig. 6G), documenting a specic eect of MoaB2
on the corresponding residues; and (iii) heights of peaks in the C-terminal half of the
σAN increased, most signicantly for residues Glu111–Asp143 (e.g., peak S131 in Fig. 6E
and peak G138 in Fig. 6F, summarized in Fig. 6H), suggesting that these residues became
more exible in the presence of MoaB2.
The fact that the addition of MoaB2 did not result in a dramatic peak broadening
shows that MoaB2 does not bind tightly to σAN. On the other hand, the specic peak
shifts document that the σAN and MoaB2 interact. In agreement with results from the
previous section, no peak of the N-terminal positively charged 60 amino-acid region is
inuenced by the strongly negatively charged MoaB2 protein. A combined chemical shift
change higher than 0.02 ppm was observed for residues between Leu92 and Ala123. The
highest values were observed in the 112AATPAVATAKAA123 stretch. Finally, a comparison
of peak broadening in the C-terminal region of the σA (not observed in the isolated free
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σAN) suggests that this region interacts with the folded domains 2, 3, and 4 of σA. In the
presence of MoaB2, this interaction is partially released and limited to residues 145–160
that show predicted helical propensity.
Taken together, both the results from NMR and pull-down experiments point to a
genuine interaction event and provide additional details about the interaction between
MoaB2 and σA, narrowing down the interaction region to the C-terminal part of σAN.
MoaB2 is not essential in vivo
To start addressing the biological role of MoaB2, we tested its essentiality in vivo, using
a CRISPR-Cas9-based system. We knocked down the expression of either moaB2 or mysA
(σA) (55). σA is essential for M. smegmatis (56) and was used as a positive control. As a
FIG 5 M. smegmatis σAN may be involved in the σA-MoaB2 interaction. (A) Schematic linear representa
tion of σA, σAΔ60aaN, σAΔN, and σB. The scale bar represents 100 amino acids (aa). σA is 466 aa long,
σAΔ60aaN is 401 aa long, σAΔN is 301 aa long, and σB is 319 aa long. (B). SDS-PAGE gel of IP of FLAG-tagged
proteins σA (LK2073), σAΔ60aaN (LK4207), and σAΔN (LK2463) using anti-FLAG antibody. The dotted line
indicates where this panel was electronically assembled from two parts of one gel. Relevant proteins are
indicated on the right side of the gel; blue arrows mark dierent σA variants; red arrows indicate MoaB2.
No FLAG strain (LK3016) was used as negative control. The identity of the bands was determined by
mass spectrometry. The experiment was performed in three biological replicates with identical results.
EXP, exponential; STA, stationary phase. (C) Relative amounts of M. smegmatis MoaB2 bound to dierent
constructs of σA in EXP and STA phase of growth, calculated from signal intensities of respective bands
from three independent SDS-PAGE gels from three independent experiments. QuantityOne (Bio-Rad)
software was used for quantication. The relative values were normalized to molecular weight of
proteins. The relative amount of σA immunoprecipitated from stationary phase (STA) was set as 1. The
bars show the average from three biological replicates, and the error bars show ±SD. P-values that are
less than 0.001 are marked as *** (t-test). The vertical arrows in the chart indicate values “zero” as the
values were even below background. (D) Comparisons of proteins associating with σA-FLAG (LK2073 over
control LK3016) vs proteins associating with σAΔN-FLAG (LK2463 over control LK3016) as determined by
quantitative LC-MS. Data from stationary phase (STA) are shown. Red spots indicate proteins signicantly
enriched (signicance: −log10 P < 2; enrichment: >log2>2). The blue spot indicates MoaB2 that was not
signicantly enriched in σAΔN-FLAG (in σA-FLAG it was signicant). Each spot is the average calculated
from three independent experiments. For values, see Supplementary Table (Table S3).
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negative control, we used a non-targeting genomic sequence single guide RNA (sgRNA).
The promoters of sgRNA and dCas9 were ATC-inducible. We positioned ATC-soaked
discs onto agar dishes where we had streaked out the respective M. smegmatis strains.
While σA depletion resulted in large growth inhibition zones, both MoaB2-depleted
and negative control strains displayed no inhibition zones (Fig. 7A). Reverse transcrip
tion-quantitative PCR (RT-qPCR) experiments conrmed that, compared to the negative
control strain, both the MoaB2 and σA mRNA had been depleted (Fig. 7B; Fig. S9).
Subsequent growth experiments then did not reveal any dierence between MoaB2
depleted and control strains (Fig. 7C). We concluded that MoaB2 was not essential, and
its depletion did not aect growth of the strain.
MoaB2 modulates transcription in vitro
Next, as MoaB2 forms a complex with σA, we decided to test whether MoaB2 is able to
aect transcription. We performed in vitro multiple-round transcriptions with puried
FIG 6 CD and 1H-15N HSQC NMR spectra of σA and in of σA with MoaB2. (A) CD spectra of σ1.1 from B. subtilis and σAN from M. smegmatis. The spectrum of B.
subtilis σ1.1 domain (residues 1–72 of σ1.1 and additional eight residues of a His-tag) manifests a shape that is typical for well-ordered protein with high α-helical
content, whereas the shape of M. smegmatis σAN (residues 1–165 of σAN preceded by a glycine) spectrum reveals a mostly disordered protein. (B) 1H-15N HSQC
NMR spectra of M. smegmatis σAN (black contours) and full-length σA (light blue contours). The narrow dispersion of proton chemical shifts indicates a disordered
protein since most of the peaks are clustered around the center of the measured spectrum and is not dispersed as is typical for proteins with well-dened
structure. (C–F) Selected regions of 1H-15N HSQC NMR spectra of M. smegmatis full-length σA. Spectra of free σA and σA with MoaB2 are shown in light blue and
red, respectively. Peaks are labeled with single-letter symbols and residue numbers. (G) Plot of the combined chemical shift changes upon MoaB2 addition.
(H) Plot of the relative heights of peaks of free σA (blue) and σA with MoaB2 (red).
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RNAP, σA or σB and MoaB2, initiating from the rRNA PrrnAPCL1 promoter. Transcrip
tion factors RbpA and CarD, which are important also for both σA- and σB-dependent
transcription, were included (28). Figure 8 then shows that increasing amounts of MoaB2
displayed progressively increasing inhibitory eects on σA-dependent transcription,
suggesting that the formation of the MoaB2-σA complex competes with the formation
of the holoenzyme (lanes 1–3). Consistent with this model, an increase in the σA amount
ameliorated the inhibitory eect of MoaB2 (lanes 4–6). Finally, transcriptions with RNAP
associated with σB were not aected by the presence of MoaB2 (lanes 7–9), consistent
with the lack of interaction between MoaB2 and σB.
We concluded that MoaB2 has the potential to modulate σA-dependent but not
σB-dependent transcription.
MoaB2 may aect the stability of σA in vivo
Finally, we speculated that MoaB2 might have additional role(s) in σA regulation. We
tested the hypothesis that MoaB2 aects the stability of σA in the cell. We compared
MoaB2 depleted (by CRISPR Cas9) and control strains. We grew the cells to mid-exponen
tial phase and blocked translation by the addition of streptomycin, which binds to the
30S ribosomal subunit and interferes with the complex formation between mRNA in the
ribosome. We then determined the relative levels of σA and the β subunit of RNAP in
both strains before and at three time points after the addition of streptomycin. Figure
S10 shows that, in the control strain, the σA level did not decrease during the time course
of the experiment, whereas in the MoaB2 depleted strain, the σA level decreased by the
4 h time point approximately twofold. The control protein, the β subunit of RNAP, was
FIG 7 M. smegmatis MoaB2 is not essential. (A) The essentiality of the M. smegmatis MoaB2-encoding gene was tested by
a CRISPR-based depletion approach. Discs soaked with ATC were placed on agar media. ATC induced expression of sgRNA
and dCas9 to deplete σA (mysA, MSMEG_2758, and LK2203) mRNA, moaB2 (MSMEG_5485 and LK2263) mRNA, and no-target
negative control (LK2261). The genes that were targeted by sgRNAs are indicated above the dishes. The experiment was
performed in three biological replicates with the same result. (B) RT-qPCR relative quantitation of three mRNAs (moaB2, mysA
and rpoC) in the moaB2 CRISPR depletion strain (LK2263; gray bars) used in (A) compared to the control strain (LK2261; black
bars, set as 1). The mRNA levels were also normalized to an external spike (RNA control introduced during the RNA extraction
protocol). mysA: codes for σA; rpoC: codes for the β′ subunit of RNAP. The graph shows averages from three independent
experiments, and error bars show ±SD. (C) Growth curves in the 7H9 rich medium of the control strain (control oligo, LK2261)
and the strain with depleted moaB2 (LK2263). The graph shows averages from three independent experiments, and the error
bars indicate ±SD. CRISPR-based depletion of MoaB2 was induced with ATC (100 ng/mL), which was added at the beginning of
growth.
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then about equally stable in both control and MoaB2-depleted strains. We note, however,
that the MoaB2-σA interaction is more prominent in stationary phase. We attempted to
determine the stability of σA also in this phase, but due to its relatively low level, we were
unable to obtain reliable data. Hence, we concluded that MoaB2 might have a stabilizing
eect on σA, but the presented experiments are not fully conclusive.
DISCUSSION
In this study, we have identied a new specic binding partner of M. smegmatis
σA, MoaB2, that appears not to interact with alternative σ factors. A recent study
also detected MoaB2 interacting with σA from Corynebacterium glutamicum (57) but
without further characterization. In our study, we further revealed the importance of
the N-terminal domain of σA for the interaction and experimentally demonstrated the
previously proposed unstructured character of this domain. We solved the 3D structure
of MoaB2 by X-ray crystallography and performed functional characterization of the
protein with respect to transcription, detecting its potential to modulate the process by
sequestering σA and aecting its stability in the cell (Fig. 9).
σA-MoaB2 interaction
Unlike E. coli σ701.1, M. smegmatis σAN has never been detected in the RNAP active site
cleft. However, due to the exible character of this domain, it cannot be excluded that
FIG 8 M. smegmatis MoaB2 modulates σA-dependent but not σB-dependent transcription in vitro.
Multiple-round transcriptions were performed with RNAP (LK1853) reconstituted with σA (LK2832) at
the 1:5 and 1:20 ratios or σB (LK1248) at the 1:5 ratio in the absence or presence of increasing amounts
of MoaB2 (LK2936) and at the presence of CarD (LK3209) and RbpA (LK3210; indicated below the graph).
Representative primary data (full gels are shown in Fig. S13B) with lane numbers are shown above the
graphs (M, MoaB2). All samples were run on 7% polyacrylamide gel (for more details see Materials and
Methods). The stochiometric amounts of MoaB2 refer to monomers. As promoter, the M. smegmatis rRNA
promoter PrrnAPCL1 was used in all panels (LK1548; full sequence is shown in Fig. S13C). All graphs show
averages from three independent experiments, and the error bars indicate ±SD. Representative gel with
transcription from the vector containing PrrnAPCL1 and the “empty” vector (LK2385) is shown in Fig.
S13A, demonstrating the identity of the transcript.
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σAN can transiently occupy this area (58). Nevertheless, it was proposed that together
with the RNAP β2 domain and β′i1 (a lineage-specic insertion in β′) σAN may restrict the
entry of DNA into the active site cleft, thereby playing a role in the formation of the open
complex, an important kinetic intermediate during transcription initiation (19).
Our data show that σAN is involved in binding of MoaB2 to σA. Surprisingly, the
60 N-terminal, predominantly positively charged aa residues of σA (Fig. 5A) do not
seem to play a role in the interaction, despite the predominantly negative surface
electrostatic potential of MoaB2 (Fig. S7). Rather, the 112AATPAVATAKAA123 stretch, which
is in the middle of an otherwise strongly acidic region between Pro61 and Pro130,
seems to mediate the interaction (Fig. 6). It is apparent, however, that the interacting
residues within this patch remain disordered. Possible interactions of other σA domains
were not characterized at the levels of individual residues due to the size limit of the
NMR experiment. Nevertheless, broadening of side-chain peaks of the σA well-ordered
domains conrmed that these domains are involved in the complex with MoaB2.
Our analytical ultracentrifugation (AUC) experiments then revealed a relative 1:1
stoichiometry for the interaction between σA and MoaB2. At the excess of σA, each
MoaB2 protomer bound to one σA molecule. The estimated relatively high (submicro
molar) binding anity suggests a strong interaction in vitro. At excess of MoaB2, the
sedimentation coecient decreased, implying incomplete saturation of MoaB2 by σA.
However, such conditions are not expected in vivo due to the assumed excess of σA in the
bacterial cell.
FIG 9 Model of interplay between MoaB2, σA, and RNAP in the mycobacterial cell. A model of functional
interactions between MoaB2, σA, and RNAP is shown. Binding of MoaB2 to σA occurs at a ratio of 1:1 (one
chain of each) and has the potential to decrease the available pool of σA, likely modulating transcription
by competing with the RNAP core for σA. σA bound to MoaB2 is not able to bind to RNAP. MoaB2 by
interacting with σA may positively aect its stability.
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Involvement of MoaB proteins in Moco biosynthesis
M. smegmatis MoaB2 has two homologs in E. coli, MogA and MoaB. MogA in E. coli
serves as an adenylyltransferase that catalyzes activation of MPT for molybdenum (Mo)
insertion to form Moco (33). However, it was demonstrated that MoaB is not required
for Moco biosynthesis in E. coli despite being encoded within the largest operon,
moaABCDE, involved in this process (31, 32). This absence of enzymatic activity is due to
the presence of a glutamate instead of a catalytically important aspartate residue (Asp56
in Pyrococcus furiosus MoaB). This residue plays a key role in the coordination of Mg2+ ions
during ATP-dependent MPT adenylation (50, 59). The biological role of E. coli MoaB thus
remains enigmatic (60). Likewise, the role of M. smegmatis MoaB2 in Moco biosynthesis is
unlikely, especially when M. smegmatis MoaB2 contains serine at the respective position
(Ser51; Fig. S11).
MoaB and MogA homologs are not equally distributed among prokaryotes. In
bacteria, species with only MogA or only MoaB or both are found. In contrast, a
search in the non-redundant protein sequence database in Archaea revealed that only
a limited number of organisms contain MogA, while MoaB is found in a larger number
of organisms (46) (Fig. S11). This suggests that MoaB may have a yet unknown function
compared to MogA in archaeal organisms. To the contrary, in Eukaryotes, it appears that
most species contain MogA but not MoaB homologs.
Genomic context of moaB2
In M. smegmatis, moaB2 is the last gene transcribed in the mprA-mprB-pepD-moaB2
operon. The mprA-mprB-pepD-moaB2 locus is present and highly conserved in all
mycobacterial species including M. tuberculosis (33). MprA and MprB are proteins
of the two-component signal transduction system MprAB, where MprB is an integral-
membrane sensor kinase, and MprA is a cytoplasmically localized response regulator.
Interestingly, these two proteins were reported to play roles during the initial adaptive
response to sub-lethal rifampicin concentration in M. tuberculosis (61). Rifampicin is an
antibiotic that binds to the DNA/RNA channel of RNAP and inhibits transcription in its
early stages (62–64). pepD, the third gene in the operon, encodes an HtrA-like serine
protease, PepD, and is directly regulated by MprAB. PepD was shown to play roles in
stress response and virulence in M. tuberculosis (65, 66).
Biological role(s) of MoaB2
The genomic context of the moaB2 gene taken together with the identied MoaB2-σA
interaction, modulatory eects of MoaB2 on transcription, and its potential eects on σA
stability is indicative of its role in gene expression and stress adaptation. In stationary
phase, the ratio of MoaB2 to σA appeared to be higher (Fig. 5B and C). As the cellular
level of M. smegmatis σA is lower in stationary phase than in exponential phase (67), we
speculate that the sequestration of σA by MoaB2 may have more pronounced eects
in stationary phase where it diminishes the already critically low level of this σ factor.
The sequestration may decrease σA-dependent transcription and favor the association
of alternative σ factors with RNAP. At the same time, this sequestration might increase
the stability of σA. The eect of MoaB2 on stability of σA may then be involved in stress
situations when translation is reduced and MoaB2 protects σA against degradation (Fig.
9). When conditions improve, the stored σA is available for transcription. In addition,
indirect evidence suggesting a MoaB2 stabilizing eect on σA from M. tuberculosis was
published recently (21): after articial depletion of σA but not σB, the level of the moaB2
transcript increased, hinting at a feedback mechanism where the cell tries to counteract
the loss of σA by increasing expression of a factor that enhances its stability.
By binding to the primary σ factor, MoaB2 may be analogous to Rsd of E. coli that
binds to σ70 (68, 69). Rsd, structurally unrelated to MoaB2, aects the availability of σ70
and competition of σ factors for RNAP, especially in stationary phase (70). Although
the deletion of Rsd has only minor eects on gene expression, it was shown that its
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function is complemented by 6S RNA, a small RNA (sRNA) that binds to and sequesters
the RNAP-σ70 holoenzyme. Rsd also modulates expression of 6S RNA, and this crosstalk
facilitates the activity of σ38 (71), a stress σ factor in E. coli (72).
In mycobacteria, no 6S RNA but Ms1 sRNA is found. Ms1, unlike 6S RNA, binds to the
RNAP core and not the primary σ factor-containing holoenzyme (67, 73). Deletion of the
Ms1-encoding gene aects the levels of RNAP (74). It will be of interest to determine
the genome-wide eects of MoaB2 (sequestration of σA) on gene expression under
normal and stress conditions and also explore these eects in combination with Ms1
(sequestration of the RNAP core) and address a potential interplay between MoaB2 and
the other proteins encoded within the mprA-mprB-pepD-moaB2 operon.
To conclude, this study has identied a new binding partner of the mycobacterial
primary σ factor, σA, and established a basis for further investigation of its interaction
with the transcription machinery and eects on gene expression. We speculate that
compounds strengthening the MoaB2-σA interaction may be developed to compromise
gene expression of the bacterium.
MATERIALS AND METHODS
Bacterial strains, plasmids, and oligonucleotides
For a detailed description of individual strains, plasmids, and oligonucleotides, see List of
strains and plasmids (Table S5) and List of oligonucleotides (Table S6) in Supplementary
Data.
Construction of M. smegmatis strains: FLAG-tagging at the native locus
σA-FLAG
The NEBuilder Assembly Tool (NEB New England Biolabs; https://nebuilder.neb.com/) was
used to design primers to create 1 × FLAG tag σA (MSMEG_2758=mysA) at its endogenous
locus (1× FLAG-tag was fused to the C-terminus of the protein). Final construct consisted
of a hygromycin resistance cassette (HYG; LK1463) anked by left and right “arms”: left
arm (LA)—approx. 500 bp long region homologous to the 3′ terminal part of the mysA
gene and containing the sequence encoding the anity tag (1× FLAG-tag: DYKDDDDK);
right arm (RA)—approx. 500 bp long region homologous to the sequence following
the 3′ end of the targeted gene. DNA fragments (LA, HYG, and RA) were amplied
by PCR with Q5 High-Fidelity DNA Polymerase (NEB) using primers #3295 + #3296,
#3297 + #3306, and #3307+#3,308 (σA-FLAG). For LA amplication, M. smegmatis mc2
155 chromosomal DNA (puried from LK3016 with ChargeSwitch gDNA Mini Bacteria Kit,
Invitrogen) was used as the template. For HYG amplication, plasmid containing HYG
(LK1463) served as the template. For RA, the respective DNA fragment was synthesized
by GeneArt Service (Invitrogen GeneArt services, ThermoFisher Scientic) and this DNA
was used for PCR amplication. Fragments were then assembled into the pUC18 plasmid
cloning vector using Gibson Assembly Cloning Kit (NEB). The mixture was transformed
into NEB 5-alpha competent E. coli cells (NEB) with the standard heat-shock transforma
tion protocol. Transformants were grown on Luria-Bertani (LB) agar supplemented with
ampicillin (100 µg/mL). This yielded construct pUC18-σA-FLAG (LK3182). The sequence
of the construct was veried by sequencing. Construct was then cleaved with restriction
enzymes BamHI/HindIII (NEB) from plasmid and transformed by electroporation into
M. smegmatis mc2 155 electrocompetent cells, prepared and performed as described
previously (75, 76), containing the pJV53/kan integrating plasmid vector (LK1321), and
plated on hygromycin (50 µg/mL) 7H10 plates (77, 78). Then, the σA-FLAG strain was
cured from pJV53 by passaging without kanamycin, resulting in the nal M. smegmatis
strain LK3207 (σA-FLAG) with 1× FLAG-tag incorporated at the native locus.
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Construction of M. smegmatis strains: FLAG-tagging at the ectopic locus
σAΔ60aaN-FLAG, σAΔN-FLAG, σB-FLAG, σE-FLAG, σF-FLAG, σG-FLAG, and σH-FLAG.
The genes coding for the σAΔ60aaN-1×FLAG (MSMEG_2758 with deletion of 60 amino
acids from the N terminus), σAΔN-3×FLAG (MSMEG_2758ΔN), σB-3×FLAG (MSMEG_2752),
σE-3×FLAG (MSMEG_5027), σF-3×FLAG (MSMEG_1804), σG-3×FLAG (MSMEG_0219), and
σH-3×FLAG (MSMEG_1914) proteins were amplied by PCR using Phusion DNA Polymer
ase (NEB) with primers #5033 + #5034 (σAΔ60aaN), #2901 + #2340 (σAΔN), #2337 + #2392
(σB), #2343 + #2395 (σE), #2345 + #2396 (σF), #2347 + #2397 (σG), and #2349 + #2398 (σH)
and M. smegmatis mc2 155 chromosomal DNA (LK3016) as the template. The 3×FLAG-tag
encoding sequence was added to respective genes in two steps. First, the gene was
amplied in the rst PCR with the reverse primer carrying part of 3×FLAG sequence,
and the rest of 3×FLAG sequence was then added in the second PCR. The two frag
ments were combined and fused by PCR with specic forward primers and the #2385
reverse primer. In the case of cloning σAΔ60aaN, 1×FLAG-tag was encoded in the reverse
primer. Subsequently, the genes were ligated into the pTetInt integrative plasmid (79)
via NdeI/HindIII or NdeI/PacI restriction sites. The constructs were veried by sequencing.
The resulting plasmids were transformed into M. smegmatis mc2 155 (LK3016) cells by
electroporation resulting in strains LK4207 (σAΔ60aaN-1×FLAG), LK2463 (σAΔN-3×FLAG),
LK2077 (σB-3×FLAG), LK2157 (σE-3×FLAG), LK2159 (σF-3×FLAG), LK2161 (σG-3×FLAG), and
LK2160 (σH-3×FLAG).
M. smegmatis containing ectopic σA-1×FLAG (LK2073) was prepared previously in our
lab (80).
Construction of E. coli strains for overexpression of σA
Gene coding for the M. smegmatis protein σA was cloned into pET28-MBP-TEV vector
(a gift from Zita Balklava & Thomas Wassmer; Addgene plasmid #69929; http://n2t.net/
addgene:69929) (81) by the method of Restriction Free PCR cloning (82). Briey, gene
for protein σA (MSMEG_2758, mysA) was amplied by PCR using Q5 High-Fidelity 2×
Master Mix (NEB) with primers #TK1+#TK2 (MSMEG_2758, mysA) from plasmid pET22b
containing cloned σA (LK1740) (80) used as the template. The cleavage site for TEV
protease was placed at the 5′ end of the gene construct. Target was amplied under
these conditions: initial denaturation (98°C, 2 min), 30 cycles of denaturation (98°C, 10 s),
annealing (65°C, 20 s), and amplication (72°C, 2 min) followed by nal extension (72°C,
5 min). Amplied gene for σA with 5′ overlapping regions from the desired insertion
sites at pET28-MBP-TEV was used as a primer for the second-cloning PCR reaction. PCR
reaction was performed in a nal volume of 20 µL containing Q5 High-Fidelity 2× Master
Mix (NEB), 20 ng pET28-MBP-TEV plasmid DNA, and 100 ng of puried amplied gene
for σA. PCR-cloning reaction was done under these conditions: single denaturation step
(98°C, 5 min), followed by 35 cycles of denaturation (98°C, 1 min), annealing (55°C, 45 s),
elongation (72°C, 8 min), and nal elongation step lasted for 10 min at 72°C. After the
parental plasmid elimination by DpnI treatment (1 µL of the enzyme to the PCR reaction
mixture), 4 µL of the PCR product was transformed to the E. coli DH5α cells. The resulting
protein fusion of MBP-σA thus has 6× His tag at the N-terminus. MBP is cleavable by
TEV protease [MBP construct end with sequence MHHHHHHVNSLEENLYFQG followed by
the second amino acid of gene MSMEG_2758 (mysA)]. The resulting construct (LK2844)
was veried by sequencing and then transformed into E. coli Lemo21 (DE3) cells (NEB)
resulting in expression strains LK2832 (σA).
Construction of E. coli strains for overexpression of MoaB2 and CarD
Gene constructs coding for the N-terminally His-tagged M. smegmatis proteins MoaB2
and CarD were amplied by PCR using Q5 High-Fidelity DNA Polymerase (NEB) with
primers #3632 + #3633 (MSMEG_5485, moaB2) and #3775 + #3776 (MSMEG_6077, carD)
from M. smegmatis mc2 155 chromosomal DNA (LK3016) as the template. The cleavage
site for TEV protease was placed at the 5′ end of the gene construct. Amplied genes
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for MoaB2 and CarD were cloned into the Champion pET302/NT-His expression vector
using the PCR by the method of Restriction Free PCR cloning (82). PCR was the same as
described in Construction of E. coli strains for overexpression of σA. Both PCR products
were separately transformed to the E. coli DH5α cells. The resulting proteins thus have
6× His-tags at their N-termini, cleavable with TEV protease [protein construct starts
with sequence MHHHHHHVNSLEENLYFQG followed by the rst amino acid of the gene
MSMEG_5485 (moaB2) or MSMEG_6077 (carD)]. The resulting constructs (LK2938–MoaB2;
LK3679–CarD) were veried by sequencing. Plasmids were then transformed into E. coli
Lemo21 (DE3) cells (LK2678) resulting in expression strains LK2936 (MoaB2) and LK3209
(CarD).
Msm RNAP-His (LK1853) and RbpA (LK3210) were prepared previously in our lab (58).
Construction of E. coli strain for overexpression of MoaB2 for crystallography
and σB
Gene construct coding for the C-terminally His-tagged M. smegmatis proteins MoaB2 and
σB were amplied by PCR using Q5 High-Fidelity DNA Polymerase (NEB) with primers
#3189 + #2472 (MSMEG_5485, moaB2) and #1153+#1154 (MSMEG_2752, σB) from M.
smegmatis mc2 155 chromosomal DNA (LK3016) as the template. Amplied genes for
MoaB2 and σB were cloned into the pET22b expression vector and transformed into E.
coli DH5α cells. The resulting constructs were veried by sequencing. Plasmids were then
transformed into E. coli BL21 (DE3) cells (LK625) resulting in expression strains LK2615
(MoaB2) and LK1248 (σB).
Media and growth conditions for M. smegmatis strains
M. smegmatis strains with “no FLAG” (wt, LK3016) and strains containing FLAG-tags at
the native loci in the genome [σA-FLAG (LK3207), RNAP-FLAG (LK2740)] were grown in
Middlebrook 7H9 medium with 0.2% glycerol and 0.05% Tween 80 at 37°C. Cells were
harvested in exponential (OD600 ∼ 0.5; 6 h of cultivation) or early stationary (OD600
∼ 2.5–3.0, 24 h of cultivation) phase of growth. When required, media were supplemen
ted with hygromycin (50 µg/mL) or streptomycin (100 µg/mL) or kanamycin (20 µg/
mL). Expression of proteins with the FLAG-tag at the ectopic locus [σA-FLAG (LK2073),
σAΔN-FLAG (LK2463), RNAP-FLAG (LK1468), σB-FLAG (LK2077), σE-FLAG (LK2157), σF-FLAG
(LK2159), σG-FLAG (LK2161), and σH-FLAG (LK2160)] was induced by ATC (10 ng/mL)
added after 3 h of growth. Cells were harvested in exponential phase as described above.
In stationary phase, ATC (10 ng/mL) for induction was added at OD600 ∼ 1.5, and cells
were harvested in early stationary phase as described above.
Media and growth conditions for E. coli strains
E. coli strains were grown in LB media at 37°C, supplemented, when needed, with
ampicillin (100 µg/mL), chloramphenicol (30 µg/mL), or kanamycin (50 µg/mL). Isopropyl
β-D-thiogalactoside (IPTG, Amresco) was added to induce expression of proteins where
indicated.
In the case of 15N-labeling for the NMR study of σAN, the E. coli strains were grown
in M9 minimal medium containing 15NH4Cl as sole source of nitrogen using standard
protocol at 37°C.
Competent E. coli strain DH5α (LK13) used for cloning, or E. coli Lemo21 (DE3) cells
(LK2678) and E. coli BL21 (DE3) cells (LK625), used for overexpression of proteins, were
prepared according to the method of (83).
Protein purication for biochemical assays
The strains of E. coli Lemo21 (DE3) cells containing expression vectors for expression of
MoaB2 (MSMEG_5485; LK2936), CarD (MSMEG_6077; LK3209), σB (MSMEG_2752; LK1248),
and RbpA (MSMEG_3858; LK3210) were grown in LB medium at 37°C until OD600 = 0.5.
Expression of MoaB2 and CarD was then induced with 800 µM IPTG and expression of
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σB and RbpA was then induced with 500 μM IPTG. Cultures after induction were grown
for 4 h at room temperature, harvested by centrifugation, and pelleted. Expression vector
pET28-MBP-TEV with σA (MSMEG_2758; LK2832) was grown in LB medium at 37°C until
the OD600 = 0.6. The growth temperature was then reduced to 20°C. After 30 min,
expression of σA was induced by the addition of 500 µM IPTG, and the culture was grown
overnight at 20°C. In the morning, cells expressing σA were harvested by centrifugation
and pelleted. Pellets of cells containing MoaB2, σA, σB, and RbpA were washed, resuspen
ded in P buer containing 300 mM NaCl, 50 mM Na2HPO4, 5% glycerol, and 3 mM
β-mercaptoethanol. In the case of CarD, T buer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5,
5% glycerol, and 3mM β-mercaptoethanol) was used during whole isolation. Pellets were
lysed using sonication [Sonopuls HD3100, Bandelin (Germany); 50% amplitude, 15 × 10 s
pulse, and 1 min pause]. Cell debris was removed by centrifugation, and supernatant
was mixed with 1 mL Ni-NTA Agarose (Qiagen) and incubated for 90 min at 4°C with
gentle shaking. Ni-NTA Agarose with bound MoaB2, CarD, σA, σB or RbpA was loaded
on a Poly-Prep Chromatography Columns (Bio-Rad), washed with P buer/T buer and
then, with P buer/T buer supplemented with 30 mM imidazole, and eluted with P
buer/T buer containing 400 mM imidazole. Fractions containing individual proteins
were pooled, based on an SDS-PAGE stained with SimplyBlue SafeStain (Invitrogen)
analysis, and dialyzed for 20 h into the cleavage buer [50 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 0.5 mM EDTA, 0.5 mM dithiothreitol (DTT), and 5% glycerol], except for σB. σB was
direclty dialyzed into the storage buer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 50 %
glycerol, 3 mM β-mercaptoethanol) and stored at –20 °C. TEV protease was then added
to dialyzed proteins (MoaB2, CarD, σA, and RbpA) at a TEV protease:protein ratio 1:20. The
cleavage was allowed to proceed for 8–12 h at 4°C. Cleaved NT-His tags and TEV protease
(His-tagged) were removed from protein solutions with Ni-NTA Agarose. One milliliter
of Ni-NTA Agarose was added to MoaB2, CarD, σA or RbpA and incubated for 90 min
at 4°C with gentle shaking. Mixtures of Ni-NTA Agarose with bound NT-His tag and
cleaved MoaB2, CarD, σA or RbpA were loaded on Poly-Prep Chromatography Columns
(Bio-Rad), and the ow through was captured. Flow through containing individual
proteins was analyzed with SDS-PAGE stained with SimplyBlue SafeStain (Invitrogen),
and individual proteins were then separately dialyzed into the starting buer (buer
A) containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 5% glycerol, and
3 mM β-mercaptoethanol for 20 h. Proteins were further puried using HiTrap Q HP
anion exchange chromatography column (Cytiva) equilibrated in starting buer (buer
A); elution saline gradient: 0.0–0.5 M of sodium chloride in the starting buer. Flow rate,
0.5 mL/min. Fractions of 0.5 mL were collected. Individual fractions from each protein
were analyzed on SDS-PAGE gel. Fractions containing pure protein were pooled and
dialyzed for 20 h into the storage buer containing 50 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 50% glycerol, and 3mM β-mercaptoethanol. Isolated proteins were stored at –20°C.
Novex Sharp Pre-stained Protein Standard (Invitrogen) was used as a protein marker on
each SDS-PAGE, unless stated otherwise.
M. smegmatis σAN (MSMEG_2758; LK2864) was expressed and puried from E. coli BL21
(DE3) with vector σAN-MBP-His, Lemo21 (DE3) as described for σA (MSMEG_2758; LK2832).
M. smegmatis RNAP was puried from E. coli BL21 (DE3) containing plasmid pRMS4
(LK1853) as described previously (58).
CRISPR Cas9 knockdown strains preparation
SgRNA oligonucleotides #2484 + #2485 targeting MSMEG_5485 (moaB2) and sgRNA
oligonucleotides #2455 + #2456 targeting MSMEG_2758 (mysA, σA) were designed
and cloned into pLJR962 according to the published protocol (55). Sequence-veried
constructs were electroporated into M. smegmatis mc2 155 (LK3016) resulting in strains
LK2263 (MSMEG_5485, moaB2 knockdown) and LK2203 (MSMEG_2758, σA knockdown).
Strain containing CRISPR Cas9 negative control, non-targeting control sgRNA (LK2261),
was used previously in our lab (74).
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Growth conditions of CRISPR Cas9 knockdown strains
CRISPR Cas9 knockdown strains [negative non-targeting control sgRNA (LK2261), moaB2
knockdown (LK2263), and σA knockdown (LK2203)] for determination of essentiality of
the genes were grown to mid-exponential phase and plated onto 7H10 solid media with
or without discs soaked with ATC (100 ng/mL). Plates were incubated at 37°C for 3–4
days, visually monitored.
CRISPR Cas9 knockdown strains [negative control non-targeting control sgRNA
(LK2261), moaB2 knockdown (LK2263)] were grown at 37°C in Middlebrook 7H9 medium
with 0.2% glycerol, 0.05% Tween 80, and ATC (100 ng/mL). For growth curves and for RNA
isolation from stationary phase, cells were grown for 24 h; OD600 was measured each
hour between 0–6 h and after 24 h. An aliquot of 25 mL of cells from stationary phase
was then cooled on ice, pelleted, and immediately frozen at –80°C. For western blot
analysis, cells were grown until mid-exponential phase (OD600 ∼ 0.6; 8 h of cultivation)
when streptomycin (100 µg/mL) was added to stop translation. Aliquots of 20 mL were
harvested at time 0 (just before the addition of streptomycin) and 4 h after the addition
of the streptomycin, pelleted, and immediately frozen at –80°C.
RNA isolation
Prior to RNA extraction from strains LK2263 (moaB2 knockdown) and LK2203 (σA
knockdown), an RNA Spike [recovery marker: Plat mRNA (718 nt) from M. musculus
prepared from pJET_Plat_IVTs; the sequence of the fragment is in Fig. S12] was added,
and the amount of which was calculated based on cell density (1.86 ng was added to
30 mL of culture at OD600 = 0.5). The RNA spike was a kind gift from Dr. Radek Malík, IMG,
Prague. Total RNA was then extracted by resuspending the pellets in 240 µL TE (pH 8.0)
plus 60 µL lysis buer (50 mM Tris-HCl, pH 8.0, 500 mM LiCl, 50 mM EDTA, pH 8.0, and
5% SDS) and 600 µL of acidic phenol (pH ~ 3):chloroform (1:1). Lysates were sonicated for
1 min on ice in fume hood and centrifuged. The aqueous phase was extracted two more
times with acidic phenol (pH ~ 3):chloroform (1:1) and precipitated with ethanol. RNA
was dissolved in water, treated with DNase (TURBO DNA-free Kit, Ambion) and reverse
transcribed into cDNA (SuperScriptIII, Invitrogen) using random hexamers.
Reverse transcription-quantitative PCR
RT-qPCR was used to amplify cDNA in duplicate reactions containing LightCycler 480
SYBR Green I Master and 0.5 µM primers (each). Primers #2476 + #2477 for moaB2
(MSMEG_5485) were designed with the Primer3 software. Primers for σA (MSMEG_2758,
mysA) #987 + #988 and rpoC (MSMEG_1368) #989 + #990 were prepared and used
previously (74). Primers #3542 + #3543 were used for the RNA spike. Negative controls
(no template reactions and reactions with RNA without reverse transcription) were run
in each experiment. For RT-qPCR, a LightCycler 480 System (Roche Applied Science) was
used. Quality of qPCR products was determined by dissociation curve analysis and the
eciency of the primers determined by standard curves. The mRNA level was quantied
on the basis of the threshold cycle (Ct) for each PCR product that was normalized to the
spike recovery marker value according to the formula 2[Ct (spike)-Ct (mRNA)].
Immunoprecipitation
Cells were grown as described in Media and growth conditions for M. smegmatis strains.
150 mL of exponential and 50 mL of early stationary phase cells were pelleted. Pellets
were resuspended in 4 mL of Lysis buer (20 mM Tris-HCl, pH 8.0, 150 mM KCl, and
1 mM MgCl2) containing 1 mM DTT, 0.5 mM phenylmethylsulfonyl uoride (PMSF), and
protease inhibitor cocktail P8849 (Sigma-Aldrich, 5 µL/mL) and sonicated 15 × 10 s
with 1 min pauses on ice and centrifuged. One milliliter of stationary and 1.5 mL of
exponential phase cells lysates were incubated overnight at 4°C with 25 µL of anti-FLAG
M2 Anity Agarose Gel (Sigma, A2220). Agarose gel beads with the captured protein
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complexes were washed 4× with 0.5 mL of lysis buer. FLAG-tagged proteins were eluted
with 60 µL of 3 × FLAG Peptide [Sigma F4799; diluted in Tris-buered saline (TBS) to
a nal concentration of 150 ng/mL]. Ten microliters of each elution was resolved on
SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen).
For immunoprecipitation of σA using antibody against σ70 (anti-σ70, clone name 2G10,
BioLegend, cat. No. 663208), M. smegmatis stationary phase cells (wt, LK3016) were
pelleted and resuspended in 20 mM Tris-HCl pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM
DTT, 0.5 mM PMSF and protease inhibitor cocktail P8849 (Sigma-Aldrich, 5 µg/mL) and
sonicated 15 × 10 s with 1 min pauses on ice and then centrifuged. Subsequently, 300 µg
(protein) of cell lysates were incubated for 2 h at 4°C with 20 µL of Dynabeads Protein A
(Invitrogen) coated either with 4 µg mouse monoclonal antibody to σ70 or 10 µg mouse
nonspecic IgG (Sigma-Aldrich) used as a negative control. The captured complexes
were washed 4× with 20 mM Tris-HCl pH 8, 150 mM KCl, and 1 mM MgCl2. The beads
were incubated in SDS sample buer for 5 min at 95°C, and eluted proteins were resolved
on SDS-PAGE gel and stained with SimplyBlue SafeStain (Invitrogen).
LC-MS/MS analysis
After immunoprecipitation, proteins were digested with 0.1 µg of trypsin solution in 50
mM ammonium bicarbonate at 37°C for 16 h. The resulting peptides were separated on
an UltiMate 3,000 RSLCnano system (Thermo Fisher Scientic) coupled to an Orbitrap
Fusion Lumos mass spectrometer (Thermo Fisher Scientic). The peptides were trapped
and desalted with 2% acetonitrile in 0.1% formic acid at a ow rate of 30 µL/min on
an Acclaim PepMap100 column [5 µm, 5 mm by 300 µm internal diameter (ID); Thermo
Fisher Scientic]. The eluted peptides were separated using an Acclaim PepMap100
analytical column (2 µm, 50 cm by 75 µm ID, ThermoFisher Scientic). The 125 min
elution gradient at a constant ow rate of 300 nL/min was set to 5% of phase B (0.1%
of formic acid in 99.9% of acetonitrile) and 95% of phase A (0.1% of formic acid) for
1 min, after which the content of acetonitrile was gradually increased. The Orbitrap
mass range was set from m/z 350 to 2,000 in the MS mode, and the instrument in
data-dependent acquisition mode acquired high-energy collisional dissociation (HCD)
fragmentation spectra for ions of m/z 100–2,000.
Protein identication and quantication
MaxQuant with Andromeda search engine (version 1.6.3.4; Max-Planck-Institute of
Biochemistry, Planegg, Germany) was utilized for peptide and protein identication
with databases of the M. smegmatis proteome (downloaded from UniProt on 20th of
December 2019) and common contaminants. Perseus software (version 1.6.2.3; Max-
Planck-Institute of Biochemistry) was used for the label-free quantication of three
biological replicates of σA-FLAG as compared to negative control (three biological
replicates of wild-type strain without any tag). The same analysis was used for the
comparison of σAΔN-FLAG as compared to negative control. The identied proteins
were ltered for contaminants and reverse hits. Proteins detected in the data were
ltered to be quantied in at least two of the triplicates in at least one condition
(557 proteins in σA-FLAG and 1,262 proteins in σAΔN-FLAG). The data were processed
to compare the abundance of individual proteins by statistical tests in the form of
student’s t-test and resulted in a volcano plot comparing the statistical signicance
(two-sided P-value) and protein-abundance dierence (fold change). Volcano plots were
created and labeled using VolcaNoseR web app (84). The mass spectrometry proteomics
data have been deposited to the ProteomeXchange Consortium (85) via the PRIDE (86)
partner repository with the data set identier PXD039468 and 10.6019/PXD039468.
Promoter DNA construction for in vitro transcription assay
The DNA fragment containing the σA-dependent promoter PrrnAPCL1 (−69/+120, +1G)
was amplied from M. smegmatis mc2 155 chromosomal DNA (LK3016) with primers
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#1474 + #1475 by PCR. The fragment was cloned into p770 [pRLG770, as described
previously (87)] using EcoRI/HindIII restriction sites (DNA sequence with annotation is
shown in Fig. S13C), transformed into E. coli DH5α competent cells, and the construct was
veried by sequencing, yielding plasmid LK1548. Supercoiled plasmid containing the
promoter was puried for in vitro transcription assays using Wizard Midiprep Purication
System (Promega) and subsequently phenol-chloroform extracted, precipitated with
ethanol, and dissolved in water.
In vitro transcription assay
In vitro transcriptions were performed with σA-dependent promoter PrrnAPCL1, an M.
smegmatis ribosomal RNA promoter (Fig. S13C) (58). Multiple-round transcription assays
were carried out essentially as described previously (88, 89) unless stated otherwise.
First, reactions were carried out in 10 µL: 250 ng of supercoiled DNA template PrrnAPCL1
(LK1548), transcription buer (40 mM Tris-HCl, pH 8.0, 10 mM MgCl2, and 1 mM DTT),
0.1 mg/mL BSA, 50 mM KCl, and NTPs [200 µM ATP and CTP; 5 mM GTP; 10 µM UTP;
2 mM of radiolabeled (α32P)-UTP]. Transcriptions were initiated by 2 µL of reconstituted
proteins [(σA ± MoaB2) + (RNAP + CarD + RbpA) for lanes 1–6 in Fig. 8 or (σB ± MoaB2) +
(RNAP + CarD + RbpA) for lanes 1–6 in Fig. 8] yielding a nal volume of 10 µL. The nal
concentrations of the reconstituted proteins in Fig. 8 and Fig. S13 were: RNAP (LK1853),
0.06 µM; σA (LK2832), 0.3 µM or 1.2 µM; MoaB2 (LK2936), 0.03 µM or 1.8 µM; σB (LK1248),
0.3 µM; CarD (LK3209), 0.3 µM; and RbpA (LK3210), 0.3 µM. Reconstitutions were carried
out for 15 min at 37°C. In vitro transcriptions were allowed to proceed for 10 min at 37°C.
Formamide stop solution (95% formamide, 20 mM EDTA, pH 8.0, 0.03% bromophenol
blue, and 0.03% xylene cyanol FF) (89) was added to stop the reaction. Samples were
denaturated for 5 min in 95°C and then loaded on polyacrylamide (PAA) gel (7% PAA,
0.1% APS, and 0.1% TEMED). Gel was run for 30 min at 30 W. Gel was dried for 30 min
at 80°C, cooled down, and exposed overnight on BAS storage phosphor screen (Fujilm).
Subsequently, the screen was scanned using Amersham Typhoon 5 Biomolecular Imager
(Cytiva) with phosphor imaging emission lter 390 BP. The signal was quantied with the
QuantityOne (Bio-Rad) software.
Western blotting
Pellets of CRISPR Cas9 knockdown strains moaB2 (LK2263) and negative control (LK2261)
were resuspended in 4 mL of Lysis buer [20 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM
MgCl2, with 1 mM DTT, 0.5 mM PMSF, protease inhibitor cocktail P8849 (Sigma-Aldrich,
5 µg/mL)] and sonicated 3 × 1 min [50% amplitude, Sonopuls HD3100, Bandelin
(Germany)] on ice and centrifuged. Concentrations of proteins in lysates were meas
ured using Bradford or Qubit 4 Fluorometer (ThermoFisher Scientic). Equal amounts
of proteins (30 µg/µL) were resolved by SDS-PAGE and detected by western blotting
on nitrocellulose membrane (Amersham Protran Premium 0.45 NC) in western blot
buer (25 mM Tris-HCl, pH 8.0, 195 mM glycine, and 20% methanol). Membrane was
blocked in 1×TBS-T (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) with
5% BSA for 1 h at 23°C and cut into two parts based on Novex Sharp Pre-stained
Protein Standard (Invitrogen) at 80 kDa. Membranes were separately incubated with
the primary antibody solution using either mouse monoclonal antibody against the β
subunit of RNAP (clone name 8RB13, BioLegend, cat. No. 663903, dilution 1:1,000) or
mouse monoclonal antibody against E. coli σ70 (clone name 2G10, BioLegend, cat. No.
663208, dilution 1:100, 5 µg/mL) overnight at 4°C. This antibody recognizes also M.
smegmatis σA (90). Subsequently, membranes were washed in 1×TBS-T and placed in
the secondary antibody solution using goat-anti-mouse IgG IR800 uorescent antibody
(WesternBright MCF-IR uorescent Western blotting kit, cat. No. K-12022–010, dilution
1:10,000) for 1 h at 23°C. Membranes were washed in 1×TBS (without Tween 20), dried for
few minutes, and scanned using Amersham Typhoon 5 Biomolecular Imager (Cytiva), IR
long emission lter at 800 nm excitation. The signal was quantied with the QuantityOne
(Bio-Rad) software.
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Circular dichroism spectroscopy
CD spectra were recorded on Jasco J-815 spectrometer (Jasco, Hachioji, Japan) at 27°C
in a 0.1 cm path-length quartz cuvette. Analyzed samples contained 7.0 µM σAN from M.
smegmatis and σA1.1 from B. subtilis [LK1345; expressed and puried as published earlier
(18)] in a buer composed of 20 mM sodium phosphate, 10 mM sodium uoride, and
pH 8.0. CD spectra were recorded from 185 to 260 nm in 15 accumulations after which
the spectra were averaged. All experiments were measured with a scanning speed of
20 nm/min, data interval of 1 nm, 1 s response time, and 1nm bandwidth.
Protein crystallization
The strain of E. coli BL21 (DE3) containing expression vector for expression of MoaB2
(MSMEG_5485; LK2615) was grown and isolated the same as described in section Protein
purication for biochemical assays—MoaB2, σA, and CarD. After elution with P buer
containing 400 mM imidazole and pooling fractions containing puried MoaB2 based
on SDS-PAGE, protein was dialyzed into the buer containing 20 mM Tris-HCl, pH 8.0,
100 mM NaCl, and 3 mM NaN3 and concentrated using Amicon Ultra-4 centrifugation
unit (Millipore). Before crystallization, the protein was centrifuged at 10,000 rpm for
10 min at 4°C. The crystallization conditions were tested using screening solutions
purchased from Qiagen (Classics I, II, Lite and PACT Suite). Routinely, 100 nL of 5 mg/mL
M. smegmatis MoaB2 protein solution were mixed with 100 nL of screening solution
applying the vapor diusion technique in 96-well Swissci triple-drop plates (sitting drop).
Screens were set up using a Phoenix (Art Robbins) robot. Plates were sealed with a
clear seal lm and incubated at 20°C in a MinstrelHT + UV storage and imaging system
(Rigaku). After 5 days, protein crystals were observed in the conditions containing 0.2
M calcium chloride, 14% (wt/vol) PEG 400, 0.1 M sodium-HEPES, and pH 7.5 (Qiagen;
Classics Lite; Well Number 74). The crystals were extracted directly from the screen
conditions, mounted onto nylon loops (Hampton Research), and ash frozen in liquid
nitrogen with the addition of 50% glycerol cryo-protectant.
Data collection and structure determination
Diraction data were collected at the DESY beamline P14 (Hamburg, Germany) at a
wavelength of 0.9762 Å with 0.05° oscillation and 0.009 s exposure per image, in 2,400
images and with crystal to detector distance of 287.47 mm using a Dectris EIGER2 CdTe
16M detector. Diraction data were processed with MOSFLM (91) and SCALA of the CCP4
program suite (92). Data collection statistics are summarized in Supplementary Table
(Table S2). The crystal structure of M. smegmatis MoaB2 was determined by molecular
replacement with MOLREP (93) using the coordinates of M. marinum MoaB2 (PDB id:
3rfq) (44) as a search model. Structure renement was performed with REFMAC5 (94)
with 5% randomly chosen reections that were set aside to calculate Rfree. The model
was manually rened using COOT (95). Water molecules were added with COOT, and
ligand molecules were included manually. The structure was rened at 2.53 Å resolution
with nal Rwork and Rfree of 0.192 and 0.26, respectively, and was validated using the
COOT validation tools. Structural coordinates were deposited in the RCSB Protein Data
Bank (PDB id: 8byr). Renement statistics are shown in Supplementary Table (Table S2).
Size exclusion chromatography
Proteins for SEC were prepared as described earlier in “Protein purication for biochemi
cal assays” in Materials and Methods in this article.
All experiments were performed using 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT
buer, an ÄKTA Prime instrument (Amersham Bioscience, UK), and HiLoadTM Superdex
200 Increase 10/300 Gl column (GE Healthcare) calibrated using Gel Filtration Markers
Kit for Protein Molecular Weights 6,500–66,000 Da (Sigma-Aldrich, MWGF70). All SEC
experiments were run at 15°C with the ow rate of 0.6 mL/min, and all chromatograms
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December 2024 Volume 206 Issue 12 10.1128/jb.00066-24 23

were collected at 254 nm (due to the extremely low absorbance of MoaB2 at 280 nm
caused by the fact that no Trp, Tyr or Cys residues are present in it). Fractions were
analyzed on SDS-PAGE. Color Prestained Protein Standard, Broad Range (New England
Biolabs) was used as marker. SDS-PAGE was performed under reducing conditions using
NuPAGE Bis–Tris 4%–12% gradient gels and Xcell SureLock mini–cell electrophoresis
system (ThermoFisher). Electrophoresis was performed according to the manufacturer’s
instructions.
σA-MoaB2 complex was formed by mixing sample of σA (fractions from SEC of σA
eluted at 12–13.5 mL) with the excess of MoaB2 (fractions from SEC of MoaB2 eluted at
13–14.5 mL) and left to equilibrate for 1 h at room temperature.
Dynamic light scattering
The M. smegmatis MoaB2 (LK2615) protein sample at 5 mg/mL in 20 mM Tris-HCl (pH
8.0), 50 mM NaCl, and 3 mM NaN3 was incubated at room temperature for 30 min before
the experiment and then centrifuged at 10,000 rpm for 10 min at 25°C. The dynamic
light scattering experiment was performed using 10 µL of protein on a Beckman-Coulter
DelsaMAX Core at a laser power of 3% and at a temperature of 25°C, with 20 scans and a
measurement time of 3 s.
MALDI mass spectrometric identication
Coomassie blue (CBB) stained protein bands were cut out from gels, chopped into small
pieces, and destained using 50 mM 4-ethylmorpholine acetate (pH 8.1) in 50% acetoni
trile (MeCN). After the supernatant removal, the gel pieces were washed step by step
with water and MeCN and then partly dried in a SpeedVac concentrator. The proteins
were digested overnight at 37°C using sequencing grade trypsin (100 ng, Promega)
in a buer containing 25 mM 4-ethylmorpholine acetate and 5% MeCN. The resulting
peptides were extracted with 40% MeCN/0.2% TFA (triuoroacetic acid).
Prior to MALDI-MS analysis, 0.5 µL of each peptide mixture was deposited on the
MALDI plate, air-dried at room temperature, and overlaid with 0.5 µL of the matrix
solution (α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA; 5 mg/mL,
Sigma). Peptide spectra were measured on a 15T Solarix XR FT-ICR mass spectrometer
(Bruker Daltonics) in a mass range of 500–6,000 Da and calibrated externally using a
PepMix II standard (Bruker Daltonics). For protein identication, the peak lists generated
using DataAnalysis 5.0 program were searched against a merged SwissProt and NCBI
database subset of M. smegmatis proteins using in-house MASCOT v.2.6 search engine
with the following settings: peptide mass tolerance of 2 ppm, maximum of missed
cleavages set to one, and variable oxidation of methionine.
NMR spectroscopy
1H-15N HSQC (53, 54), HNCACB (96) CBCA(CO)NH (97), 15N-edited TOCSY (98), 15N-edited
NOESY (99), and 5DHN(CA)CONH (100) NMR spectra were recorded on 850 and 950 MHz
NMR Bruker NEO HD spectrometers equipped with a 5 mm triple-resonance (1H-13C-15N)
inverse cryogenic probe with cooled 1H and 13C preampliers and with z-axis gradients.
The temperature was calibrated using a standard sample of neat methanol. Spectra were
processed using NMRPipe software (101) and analyzed using SPARKY (102). The NMR
sample used for partial assignment consisted of 150 µM [13C, 15N]-σAN (residues 1–160;
LK2863), 50 mM NaPi, pH 7.0, 50 mM NaCl, 0.5 mM TCEP, 2 mM NaN3, and 10% D2O, and
the spectra were recorded at 27°C. Interactions between σA and MoaB2 were studied at
20°C using 125 µM [15N]-σA or 125 µM [15N]-σA (LK2832) and 250 µM unlabeled MoaB2
(LK2936) in 50 mM HEPES, pH 7.0, 100 mM NaCl, 0.5 mM TCEP, 2 mM NaN3, and 6% D2O.
The combined chemical shift changes were calculated as Δδ =[δH2 + (0.2δN)2]1/2, where
δH and δN are the 1H and 15N chemical shifts, respectively.
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Analytical ultracentrifugation
AUC experiments were performed using Optima AUC analytical ultracentrifuge
(Beckman Coulter) equipped with an An-50 Ti rotor. Before the experiment, σA and
MoaB2 were brought to 50 mM HEPES (pH 7.5), 100 mM NaCl, 0.5 mM TCEP, and 3 mM
NaN3 by overnight dialysis, and the buer was used as an optical reference. σA and
MoaB2 concentrations were determined using the absorbance at 280 nm (A280) and
Bradford method and validated using amino acid analysis.
SV experiments were carried out at 20°C in standard double-sector epoxy-carbon
centerpiece cells with 12 mm optical path-length (Beckman Coulter) loaded with 425 µL
of both sample and reference solution. The SV experiment was done with σA (19.4 µM)
and MoaB2 (77.6 µM) samples and a set of σA:MoaB2 mixtures (molar ratios of 1:0.1 to
1:4, all containing 19.4 µM σA). Data were collected using absorbance (280 nm) and/or
interference optical systems at a rotor speed of 48,000 rpm. The data were analyzed in
Sedt 16.1 c (103) with the c(s) distribution model. For the regularization procedure, a
condence level of 0.68 was used. The partial-specic volumes of σA and MoaB2, solvent
density, and viscosity were predicted using Sednterp 3 (104). GUSSI 2.1.0 was used for
plotting of c(s) distributions and peak integration (105).
Additionally, the MSSV approach (106) was used to determine the relative stoichiom
etry of σA and MoaB2 in the complex. The spectral decomposition was performed by
analyzing absorbance (280 nm) and interference data of the 1:0.3 σA:MoaB2 molar ratio
using the multi-wavelength discrete/continuous distribution analysis model in Sedphat
15.2b (107). Concentrations of σA and MoaB2 in the complex were determined by
integrating the peak of the complex in the ck(s) distributions.
ACKNOWLEDGMENTS
We thank Dr. Radek Malík for kindly providing plasmid for preparation of the RNA
Spike Mix and Petra Sudzinová and Jana Wiedermannová for critically reading the
manuscript. The synchrotron data wasdata were collected at P14 beamline operated
by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). We
would like to thank Dr. Gleb Bourenkov for the assistance in using the beamline.
Computational resources were provided by the ELIXIR-CZ project (LM2018131), part
of the international ELIXIR infrastructure. We acknowledge the Center of Molecular
Structure—CF Protein Production (in vitro σA production), CF Crystallization of Proteins
and Nucleic Acids and CF Structural Mass Spectrometry of CIISB (Institute of Biotechnol
ogy, Vestec, Czech Republic) and the CF Biomolecular Interactions and Crystallization
and Josef Dadok National NMR Center of CIISB (CEITEC, Masaryk University, Brno, Czech
Republic), Instruct-CZ Center, part of Instruct-ERIC, supported by MEYS CR (LM2023042
and LM2018127) and European Regional Development Fund-Project „UP CIISB" (No.
CZ.02.1.01/0.0/0.0/18_046/0015974).
The project was funded by Czech Science Foundation Grant Nos. 19–12956S and
22–12023S (to L.K. and L.Ž.), 23–05622S (to J.H.) and the project National Institute of
Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103)—Funded
by the European Union—Next-Generation EU (to L.K.) and by the institutional support of
IBT CAS, v.v.i. (RVO: 86652036).
AUTHOR AFFILIATIONS
1Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the
Czech Academy of Sciences, Prague, Czechia
2Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czechia
3Faculty of Science, National Centre for Biomolecular Research, Masaryk University, Brno,
Czechia
4Institute of Biotechnology of the Czech Academy of Sciences, Centre BIOCEV, Vestec,
Czechia
5Laboratory of Regulatory RNAs, Faculty of Science, Charles University, Prague, Czechia
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December 2024 Volume 206 Issue 12 10.1128/jb.00066-24 25

6Institute of Microbiology of the Czech Academy of Sciences, Centre BIOCEV, Vestec,
Czechia
7Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague,
Czechia
AUTHOR ORCIDs
Barbora Brezovská http://orcid.org/0009-0008-7938-1825
Jarmila Hnilicová http://orcid.org/0000-0002-0953-491X
Lukáš Žídek http://orcid.org/0000-0002-8013-0336
Libor Krásný http://orcid.org/0000-0002-4120-4347
FUNDING
Funder Grant(s) Author(s)
Czech Science Foundation 19-12956S Lukáš Žídek
Libor Krásný
Czech Science Foundation 22-12023S Lukáš Žídek
Libor Krásný
Czech Science Foundation 23-05622S Jarmila Hnilicová
MEYS, Funded by the European Union-Next
Generation EU LX22NPO5103 Libor Krásný
Czech Academy of Sciences RVO: 86652036 Jan Dohnálek
AUTHOR CONTRIBUTIONS
Barbora Brezovská, Formal analysis, Investigation, Methodology, Validation, Visualization,
Writing – original draft, Writing – review and editing | Subhash Narasimhan, Formal
analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing –
review and editing | Michaela Šiková, Investigation, Visualization, Writing – review and
editing | Hana Šanderová, Investigation, Visualization, Writing – review and editing |
Tomáš Kovaľ, Investigation, Methodology, Validation, Visualization, Writing – review and
editing | Nabajyoti Borah, Investigation, Visualization, Writing – review and editing |
Mahmoud Shoman, Investigation, Writing – review and editing | Debora Pospíšilová,
Investigation, Writing – review and editing | Viola Vaňková Hausnerová, Investigation,
Writing – review and editing | Dávid Tužinčin, Investigation, Visualization, Writing –
review and editing | Martin Černý, Investigation, Visualization, Writing – review and
editing | Jan Komárek, Investigation, Methodology, Writing – review and editing | Martina
Janoušková, Investigation, Writing – review and editing | Milada Kambová, Investiga
tion, Writing – review and editing | Petr Halada, Investigation, Methodology, Writing –
review and editing | Alena Křenková, Investigation, Methodology, Visualization, Writing
– review and editing | Martin Hubálek, Investigation, Methodology, Writing – review
and editing | Mária Trundová, Investigation, Methodology, Writing – review and editing
| Jan Dohnálek, Formal analysis, Funding acquisition, Resources, Supervision, Writing –
review and editing | Jarmila Hnilicová, Conceptualization, Funding acquisition, Investiga
tion, Resources, Writing – review and editing | Lukáš Žídek, Conceptualization, Funding
acquisition, Investigation, Resources, Supervision, Writing – original draft, Writing –
review and editing | Libor Krásný, Conceptualization, Funding acquisition, Methodology,
Resources, Supervision, Writing – original draft, Writing – review and editing
DATA AVAILABILITY
The authors declare that all data supporting the ndings of this study are available
within the paper and its supplementary information les. References to data stored in
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December 2024 Volume 206 Issue 12 10.1128/jb.00066-24 26

specic databases (PDB, PRIDE) are provided in Materials and Methods where appropri
ate.
ADDITIONAL FILES
The following material is available online.
Supplemental Material
Supplemental gures and tables (JB00066-24-s0001.pdf). Fig. S1 to Fig. S13 Tables S2
and S4 to S6.
Table S1 (JB00066-24-s0002.xlsx). Contains a list of proteins pulled down with σA-FLAG
(LK3207).
Table S3 (JB00066-24-s0003.xlsx). Contains a list of proteins pulled down with σAΔN-
FLAG (LK2463).
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