Functional Genomics Reveals Extended Roles of the Mycobacterium tuberculosis Stress Response Factor σ^H

Abstract

Mycobacterium tuberculosis is one of the most successful pathogens of humankind. During infection, M. tuberculosis must cope with and survive against a variety of different environmental conditions. Sigma factors likely facilitate the modulation of the pathogen's gene expression in response to changes in its extracellular milieu during infection. σ^H, an alternate sigma factor encoded by the M. tuberculosis genome, is induced by thiol-oxidative stress, heat shock, and phagocytosis. In response to these conditions, σ^H induces the expression of σ^B, σ^E, and the thioredoxin regulon. In order to more effectively characterize the transcriptome controlled by σ^H, we studied the long-term effects of the induction of σ^H on global transcription in M. tuberculosis. The M. tuberculosis isogenic mutant of σ^H (Δ-σ^H) is more susceptible to diamide stress than wild-type M. tuberculosis. To study the long-term effects of σ^H induction, we exposed both strains to diamide, rapidly washed it away, and resumed culturing in diamide-free medium (post-diamide stress culturing). Analysis of the effects of σ^H induction in this experiment revealed a massive temporal programming of the M. tuberculosis transcriptome. Immediately after the induction of σ^H, genes belonging to the functional categories “virulence/detoxification” and “regulatory proteins” were induced in large numbers. Fewer genes belonging to the “lipid metabolism” category were induced, while a larger number of genes belonging to this category were downregulated. σ^H caused the induction of the ATP-dependent clp proteolysis regulon, likely mediated by a transcription factor encoded by Rv2745c, several members of the mce1 virulence regulon, and the sulfate acquisition/transport network.

Tuberculosis (TB) is responsible for over 2 million deaths annually (42). The failure of the BCG vaccine (1) and the emergence of drug-resistant strains of Mycobacterium tuberculosis have worsened this situation (34). To develop effective anti-TB drugs and vaccines, we need to define the mechanisms that help establish a long-term infection by M. tuberculosis.

Sigma (σ) factors bind to the RNA polymerase and influence its promoter specificity (27). Most eubacteria contain a principal σ factor, which regulates the transcription of housekeeping genes. A variable number of alternate σ factors control responses to specific environmental stimuli, adaptation to stress, and bacterial virulence (35). The pathogenesis of TB involves multiple phases and is believed to involve a carefully deployed series of adaptive bacterial virulence factors. It is conceivable that temporal expression of specific regulons controlled by the induction or availability of one or more sigma factors (10, 13) allows M. tuberculosis to survive in multiple phases of the disease process (16). One such factor, σ^H, is induced after heat, redox, nitrosative, and acid stress (25, 30, 41, 45) and phagocytosis (16). Its activity is regulated in a redox-dependent manner by the antisigma factor RshA (50) as well as by the protein kinase PknB (38). An isogenic Δ-σ^H mutant is attenuated in a mouse model and fails to induce typical pulmonary granulomatous pathology (25). σ^H directs the transcription of 31 genes, including those which encode extracytoplasmic function σ factors σ^E and σ^B and proteins involved in the maintenance of intrabacterial reducing capacity (25, 30). Expression of σ^H is induced after entry of M. tuberculosis into the host cells (19, 45), and this state of induced σ^H expression likely persists for a long period of time. Such continuing expression of σ^H must further affect gene expression in M. tuberculosis. The regulation of other transcription factors by σ^H creates a regulatory network with a potential for downstream modulation of gene expression. Induction of these proteins must in turn have further indirect impact on M. tuberculosis gene expression as a function of time. The study of these longer-term, enduring transcriptome changes will allow us to better understand the response of M. tuberculosis to phagocytosis and infection. We therefore sought to more clearly define these enduring effects of σ^H.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Wild-type M. tuberculosis and the Δ-σ^H mutant were grown at 37°C in Middlebrook 7H9-albumin-dextrose-catalase-Tween 80-glycerol broth or 7H10-Dubos oleic acid complex-glycerol agar.

Diamide stress.

We measured the effect of diamide on the viability of wild-type M. tuberculosis and Δ-σ^H by the CFU method. Aliquots of cultures 1, 2, 3, and 4 h after addition of diamide were serially diluted and plated. These results were compared to those for the experiment where diamide was withdrawn after 1 h. Cultures grown to mid-log phase were exposed to 10 mM diamide for 1 h. The cultures were centrifuged (1,455 × g, 5 min), washed in diamide-free medium, and further cultured in an equal amount of this medium. These cultures were designated post-diamide stress (PDS) cultures. Aliquots obtained before diamide addition and at 0, 30, 60, 90, 120, and 180 min PDS were serially diluted and plated.

Isolation of RNA.

RNA was isolated using the Trizol bead beater method (15), DNase treated, and purified (RNeasy; Qiagen) and its quantity assessed using a NanoDrop spectrophotometer.

Microarray studies.

Whole-genome oligonucleotide microarrays representing ∼4,750 M. tuberculosis open reading frames were obtained from the Pathogen Functional Genomic Resource Center (PFGRC) and used as recommended (ftp://ftp.jcvi.org/pub/data/PFGRC/pdf_files/protocols/). Biological replicate hybridizations were performed using PDS samples from wild-type M. tuberculosis and the mutant. Spots with imperfect morphologies, low signal-to-noise ratios, saturated intensities, and high degrees of variability between replicates were excluded from the analysis. Data were analyzed using S⁺ (Spotfire DecisionSite; TIBCO-Spotfire, Inc.) to determine statistically significant changes in expression, with adjustment for intensity bias by locally weighted scatter plot smoothing normalization (57). Genes were considered significantly and differentially expressed if they showed >1.75-fold changes with P values of <0.05.

Reverse transcription and relative quantification of mRNA by quantitative PCR (qPCR).

Quantitative real-time reverse transcription-PCR was performed with cDNA corresponding to 50 ng RNA by using a SYBR green Supermix kit (Applied Biosystems) to interrogate the expression of σ^H, σ^E, σ^B, trxB2, hsp, ctpG, bpoB, Rv2745c, clpP1, and mce1A. Expression levels were normalized using σ^A as an invariant transcript, and the average relative expression levels were determined for four replicate experiments by using the following formula: (PDS level for wild-type M. tuberculosis − pre-diamide stress level for wild-type M. tuberculosis) − (PDS level for Δ-σ^H − pre-diamide stress level for Δ-σ^H).

Generation of rabbit polyclonal anti-Rv2745c antibody.

A predicted highly antigenic peptide sequence specific to the C terminus of the Rv2745c-encoded protein (VREVV GDVLR GARMS QGRTL REV-C) was used to immunize rabbits. The animals were boosted 4 and 6 weeks after prime immunization. Antisera were collected 2, 3, and 4 weeks after the final boost, titrated, and affinity purified. The antibody specifically recognized the product of the M. tuberculosis Rv2745c gene.

Isolation of proteins, SDS-polyacrylamide gel electrophoresis, and Western blotting.

Total protein was isolated from 50 ml of each culture by using a B-PER (Pierce) kit. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed on a 12% gel by using 20 μg protein. The membrane was incubated with anti-Rv2745c antibody (1:1,000) and then with a goat anti-rabbit antibody (1:10,000) conjugated to horseradish peroxidase (Bio-Rad).

Acetamide-induced expression of the M. tuberculosis Rv2745c gene.

The M. tuberculosis CDC1551 MT2816 (Rv2745c) gene was fused to the Mycobacterium smegmatis acetamidase promoter (P_ace) on an integrative vector, pSCW38 (22). Following transformation of M. tuberculosis, the resulting strain (M. tuberculosis::pSM12) and its vector-only control (M. tuberculosis::pSCW38) were grown to log phase and induced with acetamide for 1 h. RNAs isolated from induced and uninduced cultures were used to perform qPCR as described earlier.

Microarray data accession number.

Microarray data can be accessed from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), using the provisional accession number GPL8382.

RESULTS

Wild-type M. tuberculosis and the Δ-σ^H mutant exhibit comparable inhibition levels PDS.

σ^H protects M. tuberculosis from heat, oxidative, nitrosative, and acid stress conditions. The Δ-σ^H mutant is more susceptible to diamide, a thiol-oxidative agent, which induces the expression of σ^H (30, 41). Prolonged culturing of the mutant in the presence of diamide would therefore lead to significant viability differences relative to wild-type M. tuberculosis, thus adversely affecting a global analysis of gene expression. When cultured continuously in the presence of diamide for 4 h, the viability of the Δ-σ^H mutant was reduced by more than 50% relative to that of wild-type M. tuberculosis (Fig. 1A). Therefore, we devised an alternate strategy (PDS culturing) to study the long-term global effects of σ^H induction on the M. tuberculosis transcriptome. After 1 h, diamide was rapidly washed away and culturing resumed in normal, diamide-free medium. However, the viabilities of the two cultures remained comparable during the PDS culturing (Fig. 1B).

FIG. 1.

Wild-type M. tuberculosis and the Δ-σ^H mutant exhibit comparable inhibition levels PDS. Both wild-type M. tuberculosis and the Δ-σ^H mutant were cultured in the presence of 10 mM diamide for 1, 2, 3, and 4 h (A) and 0 h (0 min), 1 h (60 min), 2 h (120 min), and 3 h (180 min) PDS (B). Data are means of results from three independent experiments, with plating for CFU performed in quadruplicates in each experiment, and the error bars represent standard deviations.

Transcriptome comparison of wild-type M. tuberculosis and the Δ-σ^H mutant PDS.

In order to verify that the expression of σ^H was induced during PDS culturing, we analyzed the presence of σ^H-specific transcripts at all PDS time points by using qPCR. As shown in Fig. 2A, expression of σ^H was induced at 0 min PDS in wild-type M. tuberculosis relative to that in the Δ-σ^H mutant, peaked at 30 min PDS, and thereafter declined, reaching background levels by 120 min. Since the expressions of both σ^E and σ^B are known to be induced in a σ^H-dependent manner following diamide stress, we also analyzed the expressions of these genes in PDS samples. The expressions of both σ^E and σ^B were induced at 0 min PDS and remained at significantly high levels until the 30-min and 60-min PDS time points, respectively (Fig. 2A).

FIG. 2.

Analysis of the comparative transcriptome levels of wild-type M. tuberculosis and the Δ-σ^H mutant PDS. (A) The expression levels of σ^H, σ^E, and σ^B in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in PDS samples were assessed by qPCR at different time points. Values represent mean differences (n-fold) ± standard deviations of results from quadruplicate experiments. (B) A two-dimensional clustering heat map shows M. tuberculosis genes expressed in a statistically significant manner for at least one PDS time point (0, 30, 60, 90, 120, or 180 min) relative to the pre-diamide induction time point. The intensity of the red color correlates with the degree of expression in wild-type M. tuberculosis relative to the level for the Δ-σ^H mutant, while the intensity of the blue color correlates with the degree of expression in the mutant relative to the level for wild-type M. tuberculosis. For each time point, changes are shown for two distinct microarrays, each of which was hybridized to a biologically independent RNA sample, along with an average value.

Wild-type M. tuberculosis transcripts made before diamide stress and at 0, 30, 60, 90, and 120 min PDS were then directly compared to the Δ-σ^H mutant transcripts by using microarrays. Figure 2B shows a hierarchical cluster of ∼800 genes differentially expressed significantly in the two strains for at least one of the PDS time points, vis-à-vis the pretreatment control. Forty-six genes were upregulated at 0 min PDS (Table 1), most of which were previously known to be dependent on σ^H for their expression (25, 30, 41). The expression of a much larger number of genes was perturbed (∼380 induced and ∼340 repressed) at the 30-min time point (see Tables 3 and 4). These substantial changes were perhaps due to the induction of σ^B, σ^E, and several other transcription factors by σ^H. Each transcription factor in turn influences a distinct group of genes, leading to such profound global effects in gene expression. In contrast, 111 genes were induced and 120 repressed in wild-type M. tuberculosis, relative to the levels for the mutant, at 60 min PDS. The number of perturbed genes continued to decline with the passage of time. At 90 min PDS, 74 genes were induced and 32 genes repressed in wild-type M. tuberculosis relative to the levels for the mutant. At 120 min PDS, only 23 genes were induced. Clearly, σ^H continues to exert its influence on specific M. tuberculosis regulons, long after its initial induction. A detailed analysis of these interactions will enhance our understanding of the M. tuberculosis stress biology.

TABLE 1.

Genes induced in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 0 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	0 min PDS
Heat shock	Rv0384c	clpB	1.148 ± 0.043	3.760 ± 0.559	0.043
	Rv3417c	groEL1	1.051 ± 0.047	4.266 ± 0.768	0.049
	Rv3418c	groES	1.051 ± 0.047	2.562 ± 0.282	0.050
	Rv0251c	hsp	0.951 ± 0.048	2.141 ± 0.143	0.018
	Rv0350	dnaK	1.068 ± 0.004	2.902 ± 0.416	0.050
	Rv0469	dnaJ1	1.003 ± 0.026	2.607 ± 0.321	0.040
Transcription	Rv2710	sigB	0.998 ± 0.017	1.998 ± 0.159	0.042
	Rv1221	sigE	0.874 ± 0.0004	2.602 ± 0.392	0.050
	Rv3223c	sigH	0.869 ± 0.029	3.471 ± 0.502	0.045
	Rv0014c	pknB	0.926 ± 0.120	1.902 ± 0.111	0.002
	Rv1674c		1.056 ± 0.125	5.065 ± 0.181	0.003
Transport	Rv2397c	cysA1	0.944 ± 0.006	2.622 ± 0.324	0.043
	Rv2399c	cysT	0.831 ± 0.035	2.146 ± 0.229	0.043
	Rv2398c	cysW	1.110 ± 0.012	2.085 ± 0.139	0.035
	Rv1859	modC	1.208 ± 0.035	2.128 ± 0.239	0.049
	Rv0924c	mntH	0.909 ± 0.003	1.862 ± 0.196	0.046
	Rv1473		0.876 ± 0.003	1.774 ± 0.087	0.016
Detoxification	Rv2641	cadI	0.965 ± 0.016	4.139 ± 0.680	0.048
	Rv3913	trxB2	0.979 ± 0.018	4.550 ± 0.780	0.047
	Rv2899c	fdhD	1.234 ± 0.010	3.036 ± 0.310	0.039
Molybdopterin biosynthesis	Rv2453c	mobA	1.236 ± 0.025	3.301 ± 0.430	0.048
	Rv3206c	moeB1	1.095 ± 0.015	3.209 ± 0.018	0.003
Sulfate metabolism	Rv1336	cysM	0.914 ± 0.058	5.058 ± 0.173	0.006
	Rv1286	cysN	0.774 ± 0.246	3.511 ± 0.827	0.047

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 0-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 3.

Genes induced in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 30 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	30 min PDS
Heat shock	Rv0384c	clpB	1.148 ± 0.043	21.896 ± 0.344	0.004
	Rv3417c	groEL1	1.051 ± 0.020	1.831 ± 0.037	0.003
	Rv0440	groEL2	1.075 ± 0.0003	1.799 ± 0.158	0.049
	Rv3418c	groES	1.342 ± 0.003	1.680 ± 0.100	0.067
	Rv0351	grpE	1.057 ± 0.005	2.939 ± 0.074	0.008
	Rv0251c	hsp	0.951 ± 0.048	20.969 ± 0.201	0.002
	Rv0353	hspR	0.977 ± 0.007	3.491 ± 0.088	0.007
	Rv0563	htpX	0.915 ± 0.027	1.840 ± 0.005	0.005
	Rv0469	dnaJ1	1.003 ± 0.026	1.638 ± 0.030	0.002
	Rv0250c		1.352 ± 0.470	16.275 ± 1.063	0.008
Virulence/detoxification	MT1966	aceA2	1.366 ± 0.002	1.900 ± 0.065	0.025
	Rv1123c	bpoB	1.017 ± 0.060	2.404 ± 0.100	0.003
	Rv2641	cadI	0.965 ± 0.060	4.698 ± 0.464	0.026
	Rv3648c	cspA	0.954 ± 0.006	2.588 ± 0.092	0.011
	Rv3874	esxB	0.993 ± 0.035	1.799 ± 0.159	0.051
	Rv3890c	esxC	1.045 ± 0.010	1.763 ± 0.002	0.002
	Rv3891c	esxD	0.907 ± 0.011	2.786 ± 0.083	0.008
	Rv3017c	esxQ	1.061 ± 0.035	2.804 ± 0.054	0.002
	MT3105	esxS	0.855 ± 0.007	3.564 ± 0.159	0.013
	Rv0169	mce1A	0.763 ± 0.013	2.924 ± 0.010	0.0003
	Rv0170	mce1B	1.055 ± 0.015	1.766 ± 0.041	0.017
	Rv0171	mce1C	1.016 ± 0.006	2.290 ± 0.052	0.010
	Rv0172	mce1D	1.023 ± 0.009	1.780 ± 0.124	0.039
	Rv0173	mce1E (lprK)	1.110 ± 0.009	2.505 ± 0.295	0.050
	Rv0174	mce1F	1.005 ± 0.013	1.750 ± 0.020	0.010
	Rv3846	sodA	1.344 ± 0.135	3.484 ± 0.338	0.049
	Rv1471	trxB1	0.986 ± 0.019	46.508 ± 1.171	0.005
	Rv3913	trxB2	0.979 ± 0.012	21.066 ± 0.647	0.007
	Rv3914	trxC	1.214 ± 0.303	24.457 ± 0.204	0.004
	Rv2525c		1.167 ± 0.002	3.111 ± 0.172	0.020
	Rv1740		0.817 ± 0.019	2.590 ± 0.166	0.023
	Rv2595		0.795 ± 0.011	2.505 ± 0.128	0.015
	Rv2829c		1.131 ± 0.026	2.342 ± 0.039	0.012
	Rv0660c		0.757 ± 0.026	2.048 ± 0.078	0.018
	Rv2547		0.770 ± 0.015	1.908 ± 0.007	0.004
	Rv2526		0.772 ± 0.013	1.853 ± 0.038	0.032
	Rv2526		0.772 ± 0.013	1.853 ± 0.038	0.032
Transcription	Rv2745c	clgR	1.015 ± 0.061	5.495 ± 0.903	0.042
	Rv1994c	cmtR	0.929 ± 0.021	6.574 ± 0.641	0.024
	Rv1909c	furA	1.289 ± 0.059	3.610 ± 0.051	0.010
	Rv2711	ideR	1.026 ± 0.007	3.610 ± 0.114	0.009
	Rv3291c	lrpA	1.119 ± 0.016	3.072 ± 0.194	0.020
	Rv0981	mprA	1.157 ± 0.017	2.360 ± 0.016	0.0002
	Rv2710	sigB	1.255 ± 0.017	2.541 ± 0.030	0.008
	Rv3414c	sigD	1.174 ± 0.041	1.832 ± 0.015	0.019
	Rv1221	sigE	0.874 ± 0.0004	19.133 ± 0.236	0.002
	Rv3223c	sigH	0.870 ± 0.029	5.883 ± 0.013	0.0007
	Rv3911	sigM	0.994 ± 0.009	1.907 ± 0.102	0.027
	Rv3232c	pvdS	0.928 ± 0.020	2.163 ± 0.206	0.033
	Rv2358	zurR	0.736 ± 0.044	1.977 ± 0.114	0.012
	Rv1674c		1.056 ± 0.125	6.603 ± 0.400	0.021
	Rv2884		0.660 ± 0.059	4.062 ± 0.131	0.012
	Rv3295		1.017 ± 0.037	3.167 ± 0.138	0.018
	Rv3291c		1.119 ± 0.016	3.072 ± 0.194	0.020
Cell wall associated	Rv0835	lpqQ	0.944 ± 0.097	2.707 ± 0.068	0.021
	Rv0847	lpqS	1.066 ± 0.088	5.901 ± 0.423	0.023
	Rv1252c	lprE	1.591 ± 0.020	2.607 ± 0.048	0.015
	Rv1541c	lprI	0.936 ± 0.011	2.433 ± 0.038	0.004
	Rv1899c	lppD	1.310 ± 0.063	2.219 ± 0.086	0.005
	Rv2080	lppJ	1.175 ± 0.022	2.567 ± 0.057	0.012
	Rv1338	murI	0.746 ± 0.050	6.006 ± 0.378	0.018
	Rv3810	pirG	1.396 ± 0.004	2.750 ± 0.111	0.017
Transport	Rv1992c	ctpG	0.916 ± 0.060	3.355 ± 0.134	0.006
	Rv2397c	cysA1	0.944 ± 0.006	2.962 ± 0.106	0.011
	Rv2399c	cysT	0.831 ± 0.035	2.893 ± 0.192	0.024
	Rv0655	mkl	1.092 ± 0.035	2.021 ± 0.054	0.014
	Rv2400c	subI	1.386 ± 0.028	2.889 ± 0.235	0.039
	MT0955	pstS1	1.693 ± 0.007	2.485 ± 0.042	0.014
	Rv1668c		0.977 ± 0.035	2.171 ± 0.013	0.004
	Rv2477c		1.032 ± 0.035	2.067 ± 0.035	0.0006
Sulfate metabolism	Rv1286	cysCN	0.778 ± 0.246	2.924 ± 0.008	0.025
	Rv1285	cysD	1.115 ± 0.100	6.483 ± 0.162	0.001
	Rv2335	cysE	0.854 ± 0.124	1.817 ± 0.019	0.024
	Rv0848	cysK2	0.842 ± 0.072	11.028 ± 1.308	0.027
	Rv1336	cysM	0.914 ± 0.058	11.442 ± 0.094	0.0007
	Rv1335	cysO	0.925 ± 0.003	6.606 ± 0.183	0.007
Hypothetical proteins	Rv2466c		1.161 ± 0.004	39.813 ± 0.476	0.002
	Rv0140		1.362 ± 0.006	30.307 ± 6.028	0.046
	Rv3463		0.996 ± 0.016	26.869 ± 0.082	0.0005
	MT3140		1.151 ± 0.032	24.694 ± 1.428	0.013

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 30-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 4.

Genes repressed in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 30 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	30 min PDS
Lipid metabolism	Rv0973c	accA2	1.206 ± 0.105	0.265 ± 0.061	0.039
	Rv0904c	accD3	1.059 ± 0.029	0.590 ± 0.058	0.041
	Rv3252c	alkB	1.427 ± 0.029	0.213 ± 0.041	0.013
	Rv2243	fabD	0.784 ± 0.062	0.357 ± 0.056	0.002
	Rv0859	fadA	1.548 ± 0.045	0.428 ± 0.193	0.047
	Rv2590	fadD9	0.985 ± 0.008	0.498 ± 0.111	0.047
	Rv3089	fadD13	0.965 ± 0.022	0.098 ± 0.005	0.004
	Rv0244c	fadE5	0.968 ± 0.036	0.229 ± 0.009	0.014
	Rv2724c	fadE20	1.261 ± 0.024	0.475 ± 0.102	0.036
	Rv0469	umaA	1.214 ± 0.008	0.567 ± 0.095	0.030
Intermediary metabolism	Rv3086	adhD	1.421 ± 0.187	0.049 ± 0.018	0.027
	Rv3841	bfrB	0.762 ± 0.002	0.182 ± 0.132	0.050
	Rv3617	ephA	0.961 ± 0.038	0.431 ± 0.063	0.043
	Rv3854c	ethA	0.844 ± 0.019	0.097 ± 0.016	0.00008
	Rv3251c	rubA	1.003 ± 0.051	0.161 ± 0.132	0.048
	Rv3250c	rubB	1.220 ± 0.059	0.175 ± 0.085	0.031
Cell wall associated	Rv0341	iniB	0.301 ± 0.163	0.294 ± 0.134	0.050
	Rv3084	lipR	1.412 ± 0.009	0.049 ± 0.028	0.006
	Rv1217c		1.120 ± 0.009	0.273 ± 0.054	0.011
	Rv2846c		0.908 ± 0.085	0.293 ± 0.083	0.035

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 30-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

Many genes induced at 0 h PDS were known to be dependent on σ^H for their expression, e.g., the σ^H, σ^B, σ^E, trxB2, trxC, groEL1, clpB, hsp, cysA1, cysT, cysW, and Rv2466c genes (Table 1). These genes play important roles in maintaining cellular redox homeostasis, as peptidases or molecular chaperones, and in sulfate acquisition (52). Genes involved in molybdenum cofactor biosynthesis and transport and modulation of cell division were also induced. Genes with lower levels of expression in wild-type M. tuberculosis than in the mutant encode ABC transporters and DNA recombination or fatty acid biosynthesis proteins (Table 2). The expression levels of a majority of genes induced at 0 min PDS continued to be elevated at 30 min PDS, with the expression levels of σ^H, σ^E, σ^B, trxB2, clpB, and hsp peaking at this time point (Table 3). Other highly induced genes at this time point included cadI, CYP132 to CYP135A1, lipL, lpqS, nuoF to nuoK, rubA, rubB, Rv0303, Rv0331, Rv0692, Rv0790c, Rv1674c, Rv2348c, Rv2884, and Rv3753c. Many of these genes are known to be expressed under nonproliferating, microaerophilic conditions (55); starvation (5); antibiotic treatment (7); or phagocytosis (19, 45), indicating an overlap in the transcriptional programs of the pathogen in response to different stress conditions that mimic intraphagosomal stress. Genes induced in M. tuberculosis at this time were related to detoxification; stress; virulence; biosynthesis of molybdenum; transport of metal cations, RNA, sulfate/thiosulfate, and phosphate; and respiration (Table 3). The genes repressed in M. tuberculosis at this time were involved in biosynthesis of fatty acids, lipid virulence factors phiocerol mycocerosate and sulfolipid-1, polyketides, mycobactin siderophores, peptidoglycan, heme-porphyrin, and amino acids (Table 4) (56). The expression levels of the mce1 operon, heat shock proteins, sulfate transporters, the thioredoxin regulon, and Rv2745c genes continued to be elevated in M. tuberculosis at 60 min PDS (Table 5). The expression of σ^H itself continued to be elevated (2.88-fold) but declined from its peak value at 30 min. Other genes induced in M. tuberculosis were involved in mycothiol-dependent detoxification; stress; fatty acid degradation; biosynthesis of phospholipids, cobalamin, and amino acids; immune-response/virulence; protein processing; energy metabolism; and macromolecule salvage (Table 5). Genes repressed in M. tuberculosis were involved in biosynthesis of fatty acids, pthiocerol dimycocerosates, polyketides, glycoproteins, polysaccharides, proto-heme, and pantothenate and transport of phosphate, drugs, glycine betaine, sugar, and Mg²⁺ (Table 6). The genes induced at 90 min PDS (Tables 7 and 8) were involved in biosynthesis of fatty acids, polyketides, the cell wall, molybdenum, polysaccharides, and thiosulfates; detoxification; transport of asparagine, cations, sulfate, phosphate, and drugs; DNA repair; protein processing; sensory regulation; and immune response. At 120 min PDS, global changes in gene expression had, to a large extent, subsided (Tables 9 and 10). These results should be interpreted in light of the fact that diamide-dependent expression of σ^H results in induction of σ^E, σ^B, and several other transcription factors in wild-type M. tuberculosis relative to the level for the Δ-σ^H mutant.

TABLE 2.

Genes repressed in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 0 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	0 min PDS
Lipid metabolism	Rv0973c	accA2	1.206 ± 0.105	0.265 ± 0.061	0.039
	Rv0904c	accD3	1.059 ± 0.029	0.590 ± 0.058	0.041
	Rv3252c	alkB	1.427 ± 0.029	0.213 ± 0.041	0.013
	Rv2243	fabD	0.784 ± 0.062	0.357 ± 0.056	0.002
	Rv0859	fadA	1.548 ± 0.045	0.428 ± 0.193	0.047
	Rv2590	fadD9	0.985 ± 0.008	0.498 ± 0.111	0.047
	Rv3089	fadD13	0.965 ± 0.022	0.098 ± 0.005	0.004
	Rv0244c	fadE5	0.968 ± 0.036	0.229 ± 0.009	0.014
	Rv2724c	fadE20	1.261 ± 0.024	0.475 ± 0.102	0.036
	Rv0469	umaA	1.214 ± 0.008	0.567 ± 0.095	0.030
Intermediary metabolism	Rv3086	adhD	1.421 ± 0.187	0.049 ± 0.018	0.027
	Rv3841	bfrB	0.762 ± 0.002	0.182 ± 0.132	0.050
	Rv3617	ephA	0.961 ± 0.038	0.431 ± 0.063	0.043
	Rv3854c	ethA	0.844 ± 0.019	0.097 ± 0.016	0.00008
	Rv3251c	rubA	1.003 ± 0.051	0.161 ± 0.132	0.048
	Rv3250c	rubB	1.220 ± 0.059	0.175 ± 0.085	0.031
Cell wall associated	Rv0341	iniB	0.301 ± 0.163	0.294 ± 0.134	0.050
	Rv3084	lipR	1.412 ± 0.009	0.049 ± 0.028	0.006
	Rv1217c		1.120 ± 0.009	0.273 ± 0.054	0.011
	Rv2846c		0.908 ± 0.085	0.293 ± 0.083	0.035

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 0-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 5.

Genes induced in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 60 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	60 min PDS
Heat shock	Rv0384c	clpB	1.148 ± 0.043	5.072 ± 0.851	0.050
	Rv0351	grpE	1.0571 ± 0.005	3.721 ± 0.363	0.031
	Rv0353	hspR	0.977 ± 0.007	4.653 ± 0.521	0.031
	Rv0352	dnaJ1	1.003 ± 0.026	10.429 ± 1.595	0.038
	Rv2373c	dnaJ2	0.851 ± 0.011	1.992 ± 0.186	0.034
	Rv3417c	groEL1	1.051 ± 0.047	6.590 ± 1.161	0.048
	Rv0563	htpX	0.915 ± 0.027	1.987 ± 0.151	0.037
	Rv0350	dnaK	1.068 ± 0.020	4.734 ± 0.078	0.006
Virulence/detoxification	Rv0170	mce1B	1.055 ± 0.015	2.300 ± 0.233	0.039
	Rv0172	mce1D	1.023 ± 0.009	1.856 ± 0.164	0.050
	Rv0174	mce1F	1.005 ± 0.013	2.368 ± 0.168	0.025
	Rv3757c	proW	0.946 ± 0.021	1.814 ± 0.022	0.0002
	Rv3891c	esxD	0.907 ± 0.011	2.208 ± 0.311	0.050
	Rv3890c	esxC	1.045 ± 0.010	1.976 ± 0.071	0.014
	Rv3913	trxB2	0.979 ± 0.018	5.060 ± 0.319	0.005
	Rv2094c	tatA	0.979 ± 0.018	1.750 ± 0.100	0.020
Transcription	Rv0182c	sigG	1.066 ± 0.018	1.674 ± 0.018	0.003
	Rv3286c	sigF	0.287 ± 0.002	1.963 ± 0.194	0.025
	Rv1221	sigE	0.875 ± 0.0004	2.944 ± 0.317	0.034
	Rv3223c	sigH	0.869 ± 0.029	2.745 ± 0.371	0.047
	Rv2711	ideR	1.026 ± 0.007	1.957 ± 0.186	0.046
	Rv1963c	mce3R	1.078 ± 0.004	2.148 ± 0.057	0.011
	Rv3295		1.017 ± 0.037	1.887 ± 0.002	0.009
	Rv2745c		1.015 ± 0.061	3.903 ± 0.496	0.033
	Rv2884		0.659 ± 0.059	2.237 ± 0.407	0.048
	Rv1674c		1.056 ± 0.125	3.504 ± 0.412	0.048
Transport	Rv2397c	cysA1	0.944 ± 0.006	6.604 ± 1.143	0.0005
	Rv2399c	cysT	0.831 ± 0.035	4.595 ± 0.462	0.003
	Rv2398c	cysW	1.110 ± 0.012	3.995 ± 0.408	0.031
	Rv0655	mkl	1.092 ± 0.029	1.935 ± 0.036	0.038
	Rv1992c	ctpG	0.916 ± 0.060	3.354 ± 0.134	0.001
	Rv3270	ctpC	1.081 ± 0.022	2.987 ± 0.400	0.049
	Rv2400c	subI	1.386 ± 0.028	5.680 ± 0.459	0.022
Sulfate metabolism	Rv0848	cysK2	0.842 ± 0.072	2.452 ± 0.395	0.044
	Rv1336	cysM	0.914 ± 0.058	7.884 ± 1.464	0.044
Cell wall associated	Rv1338	murI	0.746 ± 0.050	2.777 ± 0.393	0.048
	Rv0847	lpqS	1.066 ± 0.088	5.469 ± 0.135	0.002
	Rv2349c	plcC	0.991 ± 0.011	1.726 ± 0.180	0.050
	Rv2051c	ppm1	1.370 ± 0.181	1.827 ± 0.274	0.045
	MT0056	ponA	0.988 ± 0.013	1.722 ± 0.172	0.047
Protein turnover	Rv0782	ptrBb	1.122 ± 0.008	1.770 ± 0.296	0.009
	Rv2110c	prcB	0.814 ± 0.034	1.624 ± 0.087	0.036
	Rv0983	pepD	1.080 ± 0.026	1.692 ± 0.416	0.029
	Rv3596c	clpC1	1.077 ± 0.015	2.553 ± 0.137	0.009
	Rv2461c	clpP1	1.118 ± 0.011	2.258 ± 0.010	0.004
	Rv2460c	clpP2	1.103 ± 0.014	2.835 ± 0.125	0.018
	Rv2457c	clpX	1.306 ± 0.016	1.901 ± 0.140	0.046
Energy metabolism	Rv3053c	nrdH	1.126 ± 0.003	1.896 ± 0.170	0.046
	Rv3146	nuoB	0.917 ± 0.013	1.998 ± 0.170	0.032
	Rv3147	nuoC	0.673 ± 0.012	2.095 ± 0.286	0.046
	Rv3150	nuoF	1.015 ± 0.031	2.011 ± 0.260	0.050
	Rv3153	nuoI	1.008 ± 0.021	2.410 ± 0.330	0.048
	Rv3154	nuoJ	0.920 ± 0.00008	1.881 ± 0.212	0.049
	Rv3155	nuoK	0.916 ± 0.005	1.860 ± 0.223	0.039
	Rv3158	nuoN	0.848 ± 0.013	1.667 ± 0.155	0.045

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 60-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 6.

Genes repressed in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 60 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	60 min PDS
Lipid metabolism	Rv0973c	accA2	1.203 ± 0.105	0.488 ± 0.023	0.040
	Rv2247	accD6	0.840 ± 0.009	0.600 ± 0.027	0.035
	Rv3252c	alkB	1.427 ± 0.029	0.250 ± 0.025	0.010
	Rv1094	desA2	0.655 ± 0.006	0.394 ± 0.062	0.048
	Rv1185c	fadD21	1.061 ± 0.057	0.504 ± 0.051	0.002
	Rv3089	fadD13	0.965 ± 0.002	0.220 ± 0.053	0.009
	Rv0404	fadD30	1.135 ± 0.045	0.555 ± 0.037	0.002
	Rv2950c	fadD29	1.051 ± 0.003	0.396 ± 0.033	0.012
	Rv2930	fadD26	1.066 ± 0.022	0.121 ± 0.043	0.012
	Rv1483	fabG1	1.788 ± 0.025	0.421 ± 0.133	0.017
	Rv2524c	fas	0.870 ± 0.047	0.419 ± 0.055	0.050
	Rv2384	mbtA	1.097 ± 0.080	0.545 ± 0.074	0.002
	Rv2382c	mbtC	0.681 ± 0.043	0.424 ± 0.007	0.042
	Rv2381c	mbtD	0.801 ± 0.040	0.375 ± 0.054	0.050
	Rv2379c	mbtF	0.854 ± 0.003	0.488 ± 0.064	0.037
	Rv2931	ppsA	0.991 ± 0.016	0.256 ± 0.100	0.036
	Rv2932	ppsB	0.808 ± 0.067	0.407 ± 0.059	0.004
	Rv2933	ppsC	1.293 ± 0.033	0.337 ± 0.072	0.024
	Rv2934	ppsD	1.303 ± 0.107	0.527 ± 0.205	0.029
	Rv2935	ppsE	0.852 ± 0.001	0.498 ± 0.076	0.047
	Rv0405	pks6	1.068 ± 0.020	0.390 ± 0.010	0.007
Information pathways	Rv0651	rplJ	1.022 ± 0.037	0.566 ± 0.095	0.028
	Rv2442c	rplU	1.051 ± 0.023	0.589 ± 0.022	0.022
	Rv1015c	rplY	0.875 ± 0.008	0.531 ± 0.008	0.011
	Rv2057c	rpmG1	1.032 ± 0.085	0.609 ± 0.016	0.036
	Rv0718	rpsH	1.117 ± 0.121	0.500 ± 0.006	0.041
	Rv2055c	rpsR2	0.814 ± 0.013	0.552 ± 0.013	0.017
	Rv0710	rpsQ	1.115 ± 0.030	0.609 ± 0.003	0.013
Transport	Rv0917	betP	0.890 ± 0.022	0.541 ± 0.083	0.039
	Rv2937	drrB	0.781 ± 0.019	0.496 ± 0.002	0.017
	Rv2938	drrC	1.059 ± 0.019	0.527 ± 0.028	0.054
	Rv2846c	efpA	0.906 ± 0.085	0.417 ± 0.085	0.0001
	Rv1811	mgtC	0.941 ± 0.031	0.635 ± 0.008	0.016
	Rv2281	pitB	1.112 ± 0.001	0.490 ± 0.057	0.020
	MT0955	pstS1	1.693 ± 0.007	0.596 ± 0.029	0.007
	Rv1217c		1.120 ± 0.009	0.608 ± 0.064	0.032
	Rv0986		0.789 ± 0.034	0.452 ± 0.109	0.050
	Rv0987		0.996 ± 0.052	0.477 ± 0.049	0.044
	Rv1349		0.904 ± 0.014	0.566 ± 0.060	0.050

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 60-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 7.

Genes induced in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 90 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	90 min PDS
Lipid metabolism	Rv3667	acs	0.921 ± 0.057	1.722 ± 0.154	0.050
	Rv0468	fadB2	1.355 ± 0.033	2.628 ± 0.040	0.007
	Rv1185c	fadD21	1.106 ± 0.057	2.284 ± 0.167	0.042
	Rv0231	fadE4	1.166 ± 0.016	1.755 ± 0.087	0.026
	Rv0242c	fabG4	1.091 ± 0.023	2.196 ± 0.076	0.010
	Rv1493	mutB	1.106 ± 0.016	1.727 ± 0.172	0.055
	Rv1182	papA3	1.265 ± 0.091	2.071 ± 0.148	0.052
	Rv2946c	pks1	0.850 ± 0.038	1.660 ± 0.225	0.051
	Rv2935	ppsE	0.852 ± 0.001	1.791 ± 0.153	0.036
Transport	Rv2920c	amt	1.029 ± 0.053	1.667 ± 0.197	0.049
	Rv2397c	cysA1	0.944 ± 0.006	2.194 ± 0.056	0.011
	Rv3117	cysA3	1.141 ± 0.014	1.702 ± 0.051	0.014
	Rv2398c	cysW	1.110 ± 0.012	1.778 ± 0.043	0.010
	Rv0908	ctpE	1.030 ± 0.033	1.978 ± 0.262	0.051
	Rv1811	mgtC	0.941 ± 0.031	1.564 ± 0.153	0.001
	Rv2281	pitB	1.112 ± 0.001	2.018 ± 0.211	0.046
	Rv1273c		0.872 ± 0.034	3.093 ± 0.576	0.055
	Rv1458c		1.119 ± 0.024	2.166 ± 0.150	0.027
Cell wall associated	Rv3793	embC	0.953 ± 0.004	1.982 ± 0.211	0.046
	Rv0399c	lpqK	0.974 ± 0.113	4.103 ± 0.553	0.047
	Rv1235	lpqY	1.080 ± 0.035	2.113 ± 0.014	0.004
	Rv1921c	lppF	1.244 ± 0.266	3.160 ± 0.567	0.035
	Rv2945c	lppX	0.783 ± 0.029	2.214 ± 0.272	0.045
	Rv0451c	mmpS4	0.893 ± 0.013	2.082 ± 0.165	0.033
	Rv2350c	plcB	0.974 ± 0.010	2.247 ± 0.272	0.045
	Rv3682	ponA2	1.135 ± 0.019	1.771 ± 0.152	0.046
	Rv0284		0.846 ± 0.015	2.213 ± 0.370	0.050
	Rv1184c		1.486 ± 0.037	3.460 ± 0.010	0.003
PE/PPE family	Rv2768c	PPE43	0.878 ± 0.049	1.850 ± 0.082	0.030
	Rv3478	PPE60	1.150 ± 0.024	1.652 ± 0.140	0.050
	Rv1172c	PE12	0.910 ± 0.0003	1.720 ± 0.174	0.047
	Rv1787	PPE25	1.128 ± 0.003	2.219 ± 0.230	0.047
	Rv2519	PE26	1.047 ± 0.052	1.643 ± 0.189	0.050
	Rv1789	PPE26	0.871 ± 0.037	1.680 ± 0.144	0.049
	Rv1430	PE16	1.013 ± 0.046	1.746 ± 0.209	0.049
	Rv3533c	PPE62	1.117 ± 0.033	2.557 ± 0.390	0.050
			0.890 ± 0.022	0.541 ± 0.083	0.039
Protein processing	Rv3596c	clpC1	1.077 ± 0.015	1.651 ± 0.026	0.016
	Rv2460c	clpP2	1.103 ± 0.014	1.838 ± 0.191	0.051
	Rv2461c	clpP1	1.118 ± 0.011	1.624 ± 0.049	0.016
Transcription	Rv3132c	devS	1.037 ± 0.069	2.556 ± 0.432	0.050
	Rv1479	moxR1	0.875 ± 0.0008	1.965 ± 0.013	0.003
	Rv2088	pknJ	0.984 ± 0.159	1.771 ± 0.326	0.047
	Rv1032c	trcS	1.012 ± 0.080	1.949 ± 0.137	0.044

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 90-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 8.

Genes repressed in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 90 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	90 min PDS
Virulence	Rv3667	hbhA	1.203 ± 0.010	0.502 ± 0.185	0.051
Lipid metabolism	Rv3825c	pks2	2.535 ± 0.003	0.550 ± 0.320	0.037
Hypothetical proteins	MT1775		1.283 ± 0.003	0.339 ± 0.117	0.031

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 90-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 9.

Genes induced in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 120 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	120 min PDS
Transport	Rv1183	mmpL10	0.911 ± 0.221	1.666 ± 0.074	0.044
	Rv0929	pstC2	1.086 ± 0.034	1.756 ± 0.059	0.031
	Rv2281	pitB	1.112 ± 0.001	1.883 ± 0.164	0.047
	Rv2397c	cysA1	0.944 ± 0.006	1.610 ± 0.148	0.050
Intermediary metabolism	Rv1318c	adc	1.130 ± 0.295	1.996 ± 0.384	0.023
	Rv3303c	lpdA	1.309 ± 0.026	1.809 ± 0.032	0.034
	Rv0557	pimB	1.385 ± 0.021	3.026 ± 0.348	0.044
	Rv0886	fprB	1.022 ± 0.077	1.841 ± 0.113	0.051
	Rv0373c	coxM	1.336 ± 0.029	3.246 ± 0.336	0.042

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 120-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script.

TABLE 10.

Genes repressed in wild-type M. tuberculosis, relative to the levels for the Δ-σ^H mutant, at 120 min PDS^a

Function	Locus tag	Gene	Fold expression		P
			Pre-diamide stress	120 min PDS
Heat shock	Rv3418c	groES	1.342 ± 0.003	0.580 ± 0.023	0.007
	Rv0440	groEL2	1.075 ± 0.0003	0.580 ± 0.0003	0.010
	Rv0251c	hsp	0.951 ± 0.048	0.613 ± 0.050	0.032
Virulence	Rv3875	esxA	1.083 ± 0.0005	0.590 ± 0.037	0.017
	Rv3874	esxB	0.993 ± 0.035	0.580 ± 0.023	0.006
	Rv0287	esxG	0.941 ± 0.018	0.580 ± 0.023	0.003
	Rv0288	esxH	1.176 ± 0.021	0.580 ± 0.021	0.016
	Rv1793	esxN	1.068 ± 0.036	0.570 ± 0.009	0.020
	Rv3019c	esxR	1.215 ± 0.018	0.586 ± 0.032	0.004
Molybdenum biosynthesis	MT3193	moaB1	1.798 ± 0.118	0.539 ± 0.111	0.001
	Rv3111	moaC1	ND	0.622 ± 0.026	0.093
	Rv3112	moaD1	ND	0.345 ± 0.067	0.023
	Rv0868c	moaD2	1.233 ± 0.024	0.565 ± 0.082	0.019

^aThe functional categorization of M. tuberculosis genes was based on their known functions as assigned by the TubercuList database (http://genolist.pasteur.fr/TubercuList/). For each gene, the changes in wild-type M. tuberculosis relative to the levels for the Δ-σ^H mutant in the pre-diamide stress samples and the 120-min-PDS samples are shown, along with standard deviations. Statistical significance was assigned using the ANOVA function within the S⁺ ArrayAnalyzer script. ND, not determined.

Verification of microarray data.

To verify our transcriptome results, we performed SYBR green qPCR assays (Fig. 3A). The expression of trxB2 was induced in wild-type M. tuberculosis relative to the level for the Δ-σ^H mutant at 0 min PDS and remained at significantly high levels at all time points. The expression of hsp was induced at 0 min PDS and peaked at 30 min PDS. The expression of ctpG was induced only at the 30-min-PDS time point. The expression of bpoB was induced at both the 30 (peak)- and the 60-min-PDS time points, though its expression had been induced only at the earlier time point in microarray studies. The Rv2745c gene was induced between 30 and 90 min PDS, peaking at the 60-min time point. The expression of clpP1 was induced as early as 0 min PDS and peaked at 60 min PDS. The expression of mce1A was induced at and peaked at 30 min PDS (Fig. 3A).

FIG. 3.

Confirmation of the PDS microarray data. (A) qPCR-based interrogation of the PDS expression levels of certain genes with perturbed expression in wild-type M. tuberculosis relative to the level for the Δ-σ^H mutant at different time points. Results are shown for trxB2, hsp, ctpG, bpoB, Rv2745c, clpP1, and mce1A. Values represent mean differences (n-fold) ± standard deviations of results from quadruplicate experiments. Statistically significant changes in expression are denoted by * (P < 0.05), ** (P < 0.01), or *** (P < 0.005). (B) Western blot of M. tuberculosis wild-type and Δ-σ^H mutant lysates obtained from cultures treated pre-diamide stress and PDS shows a σ^H-dependent expression of Rv2745c. Lysates were probed with an affinity-purified polyclonal antibody raised against an epitope on the protein encoded by the M. tuberculosis Rv2745c gene. Lane A, prestained protein ladder (Invitrogen); lane B, lysate from log-phase pre-diamide stress M. tuberculosis culture; lane C, lysate from log-phase pre-diamide stress Δ-σ^H culture; lanes D, F, H, and J, lysates from 0-, 30-, 60-, and 90-min-PDS M. tuberculosis cultures; lanes E, G, I, and K, lysates from 0-, 30-, 60-, and 90-min-PDS Δ-σ^H cultures.

A polyclonal antibody against the Rv2745c-encoded transcription factor specifically detected the induced levels of this protein at 30, 60, and 90 min PDS only in wild-type M. tuberculosis (Fig. 3B). The corresponding band was not detected in comparable samples obtained from the Δ-σ^H mutant. It has previously been reported that the expression of Rv2745c is controlled by σ^E (14, 51). The inability of the Δ-σ^H mutant to induce the expression of σ^E likely results in the lack of induction of the Rv2745c gene.

Conditional expression of the Rv2745c gene in M. tuberculosis induces the expression of the clp regulon.

The Rv2745c gene was cloned downstream of the M. smegmatis P_ace in an integrative vector and used to transform M. tuberculosis. The conditional expression of Rv2745c gene was induced in this strain by adding acetamide at log phase. As measured by qPCR, the P_ace-dependent expression of Rv2745c for 1 as well as 2 h led to high levels of induction of clpP1, clpP2, and clpC1 (Fig. 4). Such induction was not observed in an M. tuberculosis strain transformed with a control vector, conclusively showing that the expression of the M. tuberculosis clp proteolysis regulon is controlled by the Rv2745c-encoded protein ClgR.

FIG. 4.

qPCR-based analysis of the expression of clp regulon genes following acetamide-dependent induction of the Rv2745c gene in M. tuberculosis. qPCR was used to interrogate the expression levels of clpP1, clpP2, and clpC1 following induction of the Rv2745c gene. Results are shown for M. tuberculosis(pSM12) (carrying an integrative vector with Rv2745c in a transcriptional fusion with the M. smegmatis acetamidase promoter) and M. tuberculosis(pSCW38) (carrying an integration-proficient vector with the acetamidase promoter without any gene, as a control). Values represent mean differences (n-fold) ± standard deviations of results from quadruplicate experiments. Statistically significant changes in expression are denoted by * (P < 0.05), ** (P < 0.01), or *** (P < 0.005).

Functional categorization of the wild-type M. tuberculosis PDS transcriptome relative to that for the Δ-σ^H mutant.

The results obtained in this study were analyzed based on the M. tuberculosis genome functional categories. The expected number of genes in a given category was calculated for a given data set, based on the total number of genes assigned to each category in the genome. This was compared to the actual number of genes of each functional category induced or repressed at a given time point (Fig. 5A and B).

FIG. 5.

Functional characterization of the PDS microarray data. Microarray data for each time point (0, 30, 60, 90, and 120 min PDS) were analyzed based on the distribution of various functional categories as defined by the TubercuList database (http://genolist.pasteur.fr/TubercuList/) for genes that were induced (A) or repressed (B) in wild-type M. tuberculosis, relative to the level for the Δ-σ^H mutant. These results were compared to the expected distribution of different functional categories (pie on the left). The pie sections are arranged in an anticlockwise manner, beginning with a red section for category 0, followed by green and yellow sections for categories 1 and 2, respectively.

DISCUSSION

The ability of M. tuberculosis to survive within the host requires resistance to various physiological and environmental stresses. Sigma factors encode a family of transcriptional regulators necessary for such adaptation processes. Expression of sigma factors σ^B, σ^E, and σ^H has been shown to be required for the resistance of the pathogen to various environmental stresses likely to be encountered in vivo (25, 26, 29-31, 41). A subset of these sigma factors is induced during the growth of M. tuberculosis within macrophages (19) and required for virulence in animal models (2, 25, 30, 32). In this report, we shed more light on the complex transcriptional network controlled by σ^H. We interpreted these results in a global context by studying the relative abundances of genes belonging to a specific functional category. Initially (at 0 and 30 min PDS), and not unexpectedly, the expression levels of a larger-than-expected number of genes belonging to the virulence/detoxification category were upregulated in M. tuberculosis. Diamide is a thiol-oxidative agent. Detoxifying proteins Hsp, GroEL (which helps properly fold proteins) (52), ClpB and HtpX (peptidases) (52), SodA (which destroys free radicals toxic to M. tuberculosis) (11), and BpoB (which controls damaging reactive oxygen species during phagocytosis) are induced to likely help the pathogen effectively deal with the consequences of such stress. Initial oxidative damage by the host macrophages may serve as an environmental cue for M. tuberculosis to turn on the expression of key virulence genes, which may in turn allow the pathogen to invade secondary cells and adapt to the host. Virulence genes induced by M. tuberculosis immediately following diamide stress include members of the mce1 operon (49); Rv2525c, a substrate of the twin-arginine translocation system, which exports folded proteins (47); Rv1740, Rv2526, Rv2547, Rv2595, and Rv2829c, PIN domain antitoxins (3); CspA, an RNA chaperone that interacts with TA networks (58); and invasin, involved in interaction with phagocytes. However, fewer than the expected number of “lipid metabolism” genes were induced at 0 and 30 min PDS (Fig. 5A). This represents a marked shift in the metabolic profile of the pathogen immediately after phagocytosis. By silencing expensive functions at a time when it is faced with a high degree of potentially destructive stress, M. tuberculosis appears to conserve cellular energy and protect its lipids and mycolic acids from oxidative damage. Further reinforcing this point, the expression levels of more than the expected number of “lipid metabolism” genes, including those involved in mycolic acid synthesis, biogenesis of mycobactin siderophores, and lipid virulence factors, were repressed at 0 and 30 min PDS (Fig. 5B). Beyond 60 min PDS, the expression levels of more than the expected number of “intermediary metabolism” genes were upregulated in M. tuberculosis. This represents an adaptation from the initial “stress defense” mode to one where the pathogen prepares metabolites for the subsequent replication and proliferation. These genes are involved in arginine biosynthesis, macromolecular salvage, and protein processing. It is energetically more expensive for M. tuberculosis to biosynthesize l-arginine than for this organism to acquire it from the external environment (17, 54). That M. tuberculosis still synthesizes arginine reflects its inability to sequester it following oxidative stress. An increased need for protein processing and turnover may be necessitated due to damage to protein structure by oxidative stress and a subsequent inability of the heat shock network to completely restore the structures of damaged proteins. Such proteins may need to be completely degraded via the ATP-dependent clp proteolytic pathway. At 60 min PDS, the “lipid metabolism” category continued to be repressed in M. tuberculosis (Fig. 5B). The 90-min-PDS time point marked a significant shift in the pattern of wild-type M. tuberculosis gene expression relative to the level for the Δ-σ^H mutant. At both 90 and 120 min PDS, the expression levels of a large number of “lipid metabolism” and “cell wall-associated” genes were elevated. This included many genes whose expression levels were initially repressed. A larger-than-expected number of “lipid metabolism” genes were no longer repressed. This demonstrates the temporal plasticity of gene expression in M. tuberculosis in response to stress. The pathogen is able to effectively silence expensive, energy-consuming cellular processes, like fatty acid biosynthesis, immediately following oxidative stress, under conditions where lipids can also be oxidized and permanently damaged. With the passage of time, the deleterious effects of oxidative stress are nullified and M. tuberculosis resumes fatty acid biosynthesis.

Our results show that σ^H induces the expression of a large number of M. tuberculosis transcriptional regulators. This in turn reshapes global transcription in M. tuberculosis, silencing lipid and virulence factor biosynthesis until the stress has been effectively managed, while inducing the expression of key regulons involved in virulence, detoxification, and protein processing. Here, we focus our attention on a few key transcription networks controlled by σ^H.

The ATP-dependent Clp proteolysis regulon is upregulated PDS.

Clp proteases perform regulatory functions by controlling the availability of key enzymes or regulators (18). The M. tuberculosis genome encodes two proteolytic (clpP1 and clpP2) and three catalytic (clpC1, clpC2, and clpX) Clp complex subunits (10, 13). In Corynebacterium glutamicum and Streptomyces coelicolor, expression of the clp operon is regulated by a transcription factor, ClgR (clp gene regulator), a homolog of Rv2745c (4, 12). The expression of the Rv2745c gene was induced beginning at 30 min PDS (Fig. 6A). The expressions of clpP1, clpP2, clpC1 (but not clpC2), clpX, and clpS were also induced at 30 min PDS and peaked at the 60-min-PDS time point. When the M. tuberculosis CDC1551 MT2816 gene (Rv2745c) was conditionally induced, the expression levels of the clp regulon genes clpP1, clpP2, and clpC1 were highly upregulated (Fig. 4). We conclude that the Rv2745c gene product positively regulates the expression of the M. tuberculosis ATP-dependent clp proteolysis regulon. The expression of Rv2745c is dependent on σ^E (14, 51), which is itself induced in a σ^H-dependent manner upon diamide stress (25, 30) as well as in a σ^H-dependent manner under yet other stress conditions (21). At this time, we are not certain if σ^H indirectly induces Rv2745c expression via σ^E or if it can also directly initiate transcription from the Rv2745c promoter. The promoter sequences recognized by M. tuberculosis σ^H and σ^E are remarkably similar, with the exception of the 3′ position of the −35 element (51). It is conceivable that the expression of Rv2745c is induced by σ^E under certain conditions where σ^H is not induced, e.g., upon cell wall damage by SDS (28), or under conditions where neither σ^H nor σ^E is induced, e.g., upon treatment with thioridizine (N. K. Dutta, S. Mehra, and D. Kaushal, submitted for publication), and by both σ^H and σ^E during oxidative and heat stress.

FIG. 6.

Genes belonging to the ATP-dependent clp regulon, the mammalian cell entry 1 (mce1) operon, and the sulfate acquisition/activation network are expressed in a σ^H-dependent manner PDS. The expression levels of clp regulon genes clpP1, clpP2, clpC1, clpC2, clpX, and Rv2745c are presented as means of results from two independent microarray experiments (A). The expression levels of the sulfate transport/activation genes cysA1, cysA3, cysCN, cysD, cysE, cysH, cysM, and cysK2 are presented as means of results from two independent array experiments (B). The expression levels of mce1 operon genes mce1A, mce1B, mce1C, mce1D, mce1E, mce1F, and Rv0176 are presented as means of results from two independent experiments (C). Statistically significant changes in expression are denoted by * (P < 0.05), ** (P < 0.01), or *** (P < 0.005).

Regulation of sulfate transport following PDS.

Sulfate assimilation is crucial for M. tuberculosis. It is an essential bionutrient with a key role in biosynthesis of cysteine, mycothiol, and coenzyme A. Moreover, extracellular presentation of sulfated metabolites may regulate host-pathogen and cell-cell communication (6). The CysTWA SubI ABC transporter complex is responsible for the active transport of inorganic sulfate across the mycobacterial cell membrane. Once imported, sulfate is phosphorylated to adenosine-5-phosphosulfate (APS) and then to 3′-phosphoadenosine 5′-phosphosulfate (PAPS), a universal sulfate donor, in a two-step reaction catalyzed by the bifunctional enzyme encoded by cysNC. GTP hydrolysis performed by the cysD product provides energy for this energetically unfavorable reaction. Together, the cysNC- and cysD-encoded proteins form a sulfate-activating complex. The sulfate moiety in APS can be reduced to sulfite by the cysH product in a reaction catalyzed by thioredoxin. Sulfite can be further reduced to sulfide by the nirA-encoded sulfite reductase (ferredoxin-dependent nitrite reductase). Sulfide is used to biosynthesize cysteine, methionine, coenzyme A, and mycothiol. De novo synthesis of cysteine involves the generation of O-acetyl-l-serine either by the cysE product, in which case it is condensed with a reduced sulfide by-product of cysK2 or cysM, or by the cysO and moeZ gene products. The expressions of cysD and cysNC were induced in wild-type M. tuberculosis, relative to the level for Δ-σ^H, at 30 min PDS. These genes have also been induced during stationary-phase growth (19) and upon reactive nitrogen (36) or oxidative (40) stress. The expressions of cysH and nirA (Rv2391) were induced at 90 and 120 min PDS in M. tuberculosis, although the level of cysH induction was below the stringent statistical-significance levels employed by us. The cysE, cysM, cysO, and moeZ genes were also induced in M. tuberculosis following stress. These results show that an entire network of genes involved in sulfate acquisition from the environment and its reduction to biologically utilizable forms for biosynthetic processes was activated. This underscores the crucial role of sulfate metabolism in the constitution of mycobacterial proteins, enzymes, and other biologically active compounds.

Regulation of the mce1 operon.

The mce1 operon is important for the virulence of M. tuberculosis (49). The six Mce1 proteins (Mce1A to Mce1F) interact with host components during infection and influence the immune response. The M. tuberculosis mce1 mutants have aberrant inflammatory cell migration and poor granuloma formation, resulting in uncontrolled bacterial growth and rapid death of mutant-infected mice (49). Members of the mce1 operon have been shown to be coexpressed with σ^H and σ^E upon phagocytosis by human macrophages (19). In this report, we show that the expressions of several members of the mce1 regulon are induced as a result of oxidative stress. These include mce1A, mce1C, and mce1E (30 min) and mce1B and mce1D (30 and 60 min). While we do not know the identity of the factor that directly mediates the expression of the mce1 regulon following oxidative stress, it is likely that σ^H is not directly involved. The induction of σ^H following oxidative stress perhaps serves as a cue for the pathogen to induce its key virulence functions in order to disseminate to secondary cells.

Transcriptional networks in M. tuberculosis following σ^H induction.

Modulation of the gene expression of numerous transcription factors helps σ^H mediate its long-term impact. The function of the Rv2745c-encoded transcription factor has already been discussed. Other transcription factors whose expression levels were perturbed in wild-type M. tuberculosis relative to the level for the mutant include Rv0273c, Rv0324, Rv0348, Rv0465c, Rv1019, Rv1049, Rv1129c, Rv1674c, Rv1956, Rv3291c, and Rv3692. Rv3291c encodes LrpA, a feast/famine family member that is induced during anaerobic conditions (55) and nutritional starvation (5) and is critical for persistence (39). Its Escherichia coli homolog regulates metabolic pathways in response to the availability of amino acids in the external environment (8). While we do not know which M. tuberculosis genes are regulated by lrpA, a large number of genes related to amino acid metabolism were induced at 60 min PDS, following the induction of lrpA at 30 min PDS.

The Rv0324-Rv0325-Rv0326 operon is induced in enduring hypoxia, along with the σ^H/σ^E regulon (48). The Rv0347-Rv0348-Rv0349 operon is induced in vivo at the late stage of chronic TB (53). Rv0348 potentiates the expression of σ^F, which orchestrates entry into the chronic stage of TB (15). The expression of Rv1049 is induced by heat shock (52), peroxide stress (48), and starvation (5). Rv1049 is required for survival in murine macrophages (43). Rv1956 is induced by starvation (5) and encodes a possible antitoxin. Rv0465c is induced following nutritional stress (20) in a σ^E- and σ^F-dependent manner. The expressions of Rv1129c and its adjoining genes Rv1130 and Rv1131 are induced in a σ^E (29)- and mprA-dependent manner (37).

The Rv1994c (cmtR)-encoded repressor CmtR senses the critical thresholds of Cd(II) and Pb(II) (9), is induced during starvation (5) and oxidative stress (48), and is required for in vivo survival (24). We propose that diamide treatment oxidizes the numerous cysteine thiols that CmtR senses metal ions through. This derepresses the cmtR regulon, leading to the induction of transporters Rv1993 and ctpG. This allows M. tuberculosis to transport out the high levels of Cd(II) and Pb(II). CmtR is able to sense metal ions upon the restoration of redox homeostasis in M. tuberculosis, eventually repressing the Rv1993-ctpG operon. We also observed the induction of the cadI gene by diamide treatment in M. tuberculosis, in contrast to an earlier report (23). However, it must be considered that the earlier report involved the use of the H37Rv strain, not the CDC1551 strain, of M. tuberculosis. Alternatively, it is possible that a duration of diamide treatment other than 1 h or a concentration of diamide other than 10 mM was used in the previous report. The function of M. tuberculosis CadI appears to be similar to that of metallothioneins, which are low-molecular-weight, cysteine-rich proteins that protect against metal toxicity. Since diamide oxidizes cysteine residues on proteins, an increased induction of cadI may be an attempt by M. tuberculosis to overcome the decreased activity of this protein.

furA encodes a global repressor that uses Fe²⁺ as a cofactor to bind the katG operator. katG is transcribed from two promoters, one generating a bicistronic furA-katG message, expressed initially during infection, and another generating a monocistronic katG mRNA, which peaks at a later stage (33). The expression of furA was induced at 30 min PDS and then declined. The expression pattern of katG was the mirror opposite; this gene was repressed at 30 min PDS, presumably due to repression by furA, and then induced as the furA levels declined, peaking at 90 min PDS. The iron-binding repressor IdeR reduces the expression of genes involved in siderophore biosynthesis, iron storage, and transport (44), including mbtABCDEFGI, fadE14, Rv3402c, and Rv3403c. All these genes exhibited reduced expression in wild-type M. tuberculosis, relative to the level for the mutant, at 30 min PDS. The expression of ideR is induced by σ^B, which is itself induced in a σ^H-dependent manner following oxidative stress. Rv2358 encodes Zur, which regulates the homeostasis and uptake of zinc, an essential element that is included in numerous enzymes and proteins as a cofactor or a structural scaffold but can be toxic at higher concentrations (28). Rv0981 (mprA) encodes a two-component response transcriptional regulatory protein, MprA (mycobacterial persistence regulator), which is induced upon starvation and in a σ^E-dependent manner.

To summarize, we have shown that σ^H not only directs the transcription of a subset of genes involved in the maintenance of redox homeostasis and protein integrity but also induces a network of transcription factors, which further induce crucial bacterial functions, such as ATP-dependent proteolysis of specific targets, acquisition and utilization of environmental sulfate toward the biosynthesis of key mycobacterial metabolites, specific virulence- and pathogenesis-related operons that help M. tuberculosis survive and disseminate in vivo, and transcription factors that help M. tuberculosis sense and respond to the levels of various metal and trace metal ions. We demonstrate the dynamic nature of the pathogen's response to adverse environmental conditions, with the expression of genes involved in lipid biosynthesis being repressed following oxidative stress and resuming upon the removal of stress. We hope to extend these findings by interrogating their intraphagosomal and in vivo relevance.