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Journal of Bacteriology logoLink to Journal of Bacteriology
. 2017 Oct 3;199(21):e00385-17. doi: 10.1128/JB.00385-17

Characterization of a Minimal Type of Promoter Containing the −10 Element and a Guanine at the −14 or −13 Position in Mycobacteria

Yan Zhu a,c, Chunyou Mao a,c, Xingyi Ge b, Zhongwei Wang a,c, Pei Lu a, Yong Zhang a, Shiyun Chen a, Yangbo Hu a,✉
Editor: Richard L Goursed
PMCID: PMC5626959  PMID: 28784819

ABSTRACT

Three key promoter elements, i.e., −10, −35, and T−15G−14N, are recognized by the σ subunit of RNA polymerase. Among them, promoters with the −10 element and either −35 or T−15G−14N are known to initiate transcription efficiently, but recent systematic analyses have identified a large group of promoters in Mycobacterium tuberculosis that contain only a −10 consensus. How these promoters initiate transcription remains poorly understood. Here, we show that promoters containing the −10 element and an upstream G located at the −14 or −13 position can successfully initiate transcription in mycobacteria. Importantly, this new type of promoter is active in the absence of other promoter consensuses, suggesting that it is a minimal promoter type. Mutation of the upstream G in promoters decreased the efficiencies of their binding with RNA polymerase and their abilities to initiate transcription in both in vitro and in vivo analyses. A glutamic acid in σ region 3.0 is essential for recognizing G−14 and G−13 and is conserved in both principal and principal-like σ factors in mycobacteria, indicating that recognition of this minimal type of promoter might be a common mechanism for transcription initiation. Consistently, more than 70% of the identified promoters in M. tuberculosis contained G−14 or G−13 upstream of the conserved −10 element, and thousands of promoters in representative mycobacterial species have been predicted using the −10 consensus and G−14 or G−13. Altogether, our study presents a universal mechanism for transcription initiation from a minimal promoter in mycobacteria, which might also be applicable to other bacteria.

IMPORTANCE In contrast to the detailed information for recognizing classic promoters in the model organism Escherichia coli, very little is known about how transcription is initiated in the human pathogen Mycobacterium tuberculosis. In this study, we characterized a new type of promoter in mycobacteria that requires only a −10 consensus and an upstream G−14 or G−13. Residues important for recognizing the −10 element and the upstream G are conserved in σA and σB from mycobacterial species. According to such features, thousands of promoters in mycobacteria can be predicted using the −10 consensus and G−14 or G−13, which suggests that transcription from this new type of promoter might be widespread. Our findings provide insightful information for characterizing promoters in mycobacteria.

KEYWORDS: Mycobacterium, promoters, sigma factors, transcription

INTRODUCTION

Transcription performed by RNA polymerase (RNAP) is the first step in gene expression. Bacterial RNAP is composed of a multisubunit core enzyme (α2ββ′ω) and an additional factor (σ) for promoter recognition (1). The RNAP core enzyme first associates with one σ factor to form the RNAP holoenzyme, which recognizes the promoter to form the unstable “closed complex” (RPc) and then isomerizes into the stable “open complex” (RPo). After initiating transcript synthesis, RNAP moves away from the promoter to form an elongation complex in which the σ factor is no longer required (1, 2).

Recognition of promoters by σ in the RNAP holoenzyme controls the specificity of transcription. Bacteria have a single principal σ, which mainly initiates the transcription of housekeeping genes, and some may have a principal-like σ and alternative σ factors, which promote the transcription of specialized genes for stress responses (3). The principal and principal-like σ factors (namely, σ70 or σA and σS or σB) share considerable sequence similarity (4). Based on biochemical and structural studies, these σ factors can be divided into four regions (regions 1 to 4) to interact with different promoter elements.

Three known elements that are recognized by principal or principal-like σ factors in bacterial promoters are −10, −35, and T−15G−14N (5). The −10 element is conserved in the sequence T−12A−11NNNT−7 in different bacteria and is recognized by region 2 of the σ factor (6, 7). Structural analyses have revealed that A−11 and T−7 on the nontemplate strand of the −10 element are buried deep in the σ region 2 pockets in RPo to stabilize this complex (8–10). The −35 element (consensus sequence of T−35TGACA−30) is recognized by σ region 4. Unlike the widely distributed −10 element, the −35 element has been identified only in certain bacterial species (11, 12). The T−15G−14N element (herein abbreviated as TGN) has been characterized in several bacterial promoters and is recognized by a conserved histidine and nearby glutamic acid (H455 and E458 in σ70 of Escherichia coli) in σ factor region 3.0 (also known as region 2.5) (13–15). The presence of TGN matches weak conservation of the −35 element in some promoters (14, 16). Among the characterized elements, promoters that contain either of two consensus sequences (−35/−10, TGN/−10, or −35/TGN) have been documented to work efficiently in E. coli (5, 17).

Most studies concerning mechanisms for transcription initiation were performed in E. coli, and situations in Mycobacterium tuberculosis, the causative agent of tuberculosis, are less well known. A previous analysis showed that the −10 regions of identified promoters are conserved but the −35 regions are not (11); only a small number of TGN promoters have been characterized in mycobacteria (13). Two recent works systematically identified transcriptional start sites (TSSs) of M. tuberculosis under normal growth and stress conditions (18, 19). The TANNNT −10 box was found upstream of 73% of the primary TSSs, but the −35 element was not conserved (18). Approximately 7% of promoters (90 of 1,290 promoters) contain the TGN element upstream of the −10 consensus (18). Interestingly, G−14 is enriched in mycobacterial promoters but its function remains to be explored (18). Together, these observations indicate that a large number of promoters contain only the conserved −10 sequence among the characterized promoter elements. The mechanisms for transcription initiation from these promoters remain poorly understood.

In this study, we showed that the guanine at the −14 or −13 position, together with the −10 element, activates transcription initiation in mycobacteria, which defines the minimal promoter as containing the −10 element and an upstream G. Importantly, this minimal type of promoter is widespread in mycobacteria and matches more than 70% of the identified promoters in M. tuberculosis.

RESULTS

G−14 and G−13 upstream of the −10 element stimulate mycobacterial rbpAp activity.

Previously, we identified a σB-activated promoter upstream of the rbpA gene in M. tuberculosis that contained only one conserved −10 element (TATCAT) (20). A form of this rbpA promoter (named rbpAp) truncated to 6 bp upstream of the −10 element, which lacks the putative −35 element (Fig. 1A, P2), still exhibited strong activity in both mycobacterial σB-overexpressed E. coli (20) (Fig. 1B) and Mycobacterium smegmatis (Fig. 1C). Promoters with mutations in the putative −35 element (P4) or upstream sequence (P3) showed activities similar to that of the parental promoter (Fig. 1A to C). These data indicate that the activity of rbpAp is not dependent on the −35 element.

FIG 1.

FIG 1

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Activities of mycobacterial rbpAp derivatives. (A) Derivatives of rbpAp from M. tuberculosis (Mtb). The −10 element is indicated as an orange box. All numbers are indicated using the TSS as +1. (B) Promoter activities of rbpAp derivatives tested in E. coli (Ec). Ecσ represents the group without expression of σ from M. tuberculosis; ck- represents the control group carrying the promoterless lacZ. (C) Promoter activities of rbpAp derivatives tested in M. smegmatis (Ms). (D) DNA sequence alignment of rbpAp sequences from mycobacterial strains. The conserved sequence from the −13 to −15 positions was generated using WebLogo (49). (E and F) Activities of rbpAp with mutations in nucleotides −13 to −14, assessed in E. coli (E) and M. smegmatis (F). *, P < 0.05; **, P < 0.01. M. can, Mycobacterium canetti; M. tub, M. tuberculosis; M. afr, Mycobacterium africanum; M. bov, Mycobacterium bovis; M. mar, Mycobacterium marinum; M. lepr, Mycobacterium leprae; M. avi, Mycobacterium avium; M. sm, Mycobacterium smegmatis.

Sequences from −15 to −13 of rbpAps in mycobacteria are conserved as CGG (Fig. 1D) but not TGN, which is known to be located at this position in E. coli (12). It is noteworthy that replacement of either G−13 or G−14 with adenosine decreased rbpAp activity to some extent, and replacement of G−14G−13 with A−14A−13 almost abolished rbpAp activity (Fig. 1E and F). Except when a new TGN element was introduced, mutations at C−15 did not change the promoter activity noticeably, and mutations of either G−13 or G−14 to other nucleotides all decreased promoter activity (see Fig. S1A and B in the supplemental material), suggesting that either G−13 or G−14 can specifically stimulate rbpAp activity.

G−14 and G−13 are widespread in mycobacterial promoters.

To explore whether the activation by G−14 and G−13 is specific to rbpAp, we analyzed promoter elements in sequence stretches between bp −49 and +1 with respect to TSSs identified in M. tuberculosis (18, 19). Among these sequences, 3,111 of a total of 4,978 promoters contained a typical TANNNT sequence as the −10 element located 5 to 7 bp upstream of the TSSs (19), and 70.7% of these promoters contained either G−14 or G−13 or both (Fig. 2A). Similar results were also obtained for 1,178 primary promoters identified by Cortes et al. (18) (Fig. S2A).

FIG 2.

FIG 2

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Roles of G−14 and G−13 in mycobacterial promoter activities. (A) Percentages of promoters containing G−14 and/or G−13 upstream of TANNNT in identified promoters of M. tuberculosis (Mtb). Promoter sequences were analyzed according to the TSSs reported by Shell et al. (19). (B) Sequences of tested promoters. Nucleotides at the −14 and −13 positions are shown with a green background. (C to H) Activities of mycobacterial promoters with mutations at −14, −13, or both positions, tested in M. smegmatis. Mutated nucleotides are shown in red. *, P < 0.05; **, P < 0.01.

The roles of G−14 and G−13 were further analyzed in other promoters from M. tuberculosis (Fig. 2B). Mutations in either of the G residues in G−14G−13-containing promoters (Rv1494p and Rv0005p) decreased the promoter activities to some extent in M. smegmatis (Fig. 2C and D) and σB-overexpressing E. coli (Fig. S1C and D). In addition, mutation of both G−14 and G−13 dramatically decreased the activities of these two promoters, as observed in rbpAp (Fig. 2C and D). The activation effects of G−14 were further tested by introducing G−14 into G−13-containing promoters (Rv2715p and Rv0873p) (Fig. 2E and F) and mutating G−14 in G−14-containing promoters (Rv2703p and Rv3583p) (Fig. 2G and H; also see Fig. S1E and F). The roles of G−13 in the mycobacterial promoters were also confirmed, as were those of G−14 (Fig. 2E and F). The introduction of either G−13 or G−14 activated the promoters without the upstream G (Fig. S2B and C). These data suggest that activation by G−14 and G−13 is widely used in mycobacterial promoters.

G−14 and G−13 contribute to promoter recognition by the σ factor.

To determine whether the G upstream of the −10 element could directly contribute to promoter recognition by the σ factor, we performed in vitro transcription using the σB-RNAP-RbpA model from M. tuberculosis (21). For both Rv1494p and Rv0005p, specific transcripts were observed in the presence of σB. In accordance with promoter activity tests in vivo, mutation of either G−14 or G−13 decreased transcript amounts, and mutations of both almost abolished the promoter activities (Fig. 3A and B).

FIG 3.

FIG 3

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Roles of G−14 and G−13 in promoter recognition. (A and B) In vitro transcription of mycobacterial σB-RNAP from Rv1494p (A) and Rv0005p (B). The promoter fragments and RNAP proteins were from M. tuberculosis. Parental promoters contain G−14G−13. Mutated nucleotides in promoters are shown in red. Quantification of transcripts from three independent tests is shown in each bottom panel (mean ± SD). **, P < 0.01. (C) DNase I footprinting of the Rv1494p (wild-type and mutant) complex with the RNAP core, σB-RNAP. RbpA is presented in all tests. The promoter DNA was labeled on the 5′ end of the nontemplate strand. The protected DNA region is shown as light gray boxes. The dark gray box indicates the −10 element.

The roles of the upstream G in promoter recognition were further elucidated by DNase I footprinting. As shown in Fig. 3C, compared with the RNAP core enzyme, σB-RNAP protected the promoter region from −45 to +25 (Fig. 3C, top), which is a feature of the RPo (22). Mutation of G−14G−13 to A−14A−13 clearly decreased the protection of the −10 and upstream G regions (Fig. 3C, bottom), providing further evidence that G−14 and G−13 directly contribute to promoter recognition.

Residue E169 in σB region 3.0 is essential for recognizing G−14 and G−13.

Direct interaction between the σ factor and promoter elements is essential for promoter recognition (4). To determine the amino acids that are essential for recognizing G−14 and G−13, 14 sites on M. tuberculosis σB that were around the DNA binding region (9) and were not completely conserved in E. coli σ70 and σS were mutated (Fig. 4A). As shown in Fig. 4B, four mutants (M127V, G153A, S158A, and E180Q) whose mutated sites were close to the core enzyme mapped in the M. smegmatis σA-RNAP structure model (PDB accession no. 5TW1) (23) (Fig. S3A) showed higher efficiencies than wild-type σB in initiating rbpAp transcription. Whether this effect was due to an affinity change for mycobacterial σB by the E. coli RNAP core enzyme is unclear. Interestingly, one mutant (E169K in region 3.0) showed a markedly lower efficiency than did wild-type σB (Fig. 4A and B).

FIG 4.

FIG 4

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Evidence that E169 in region 3.0 of M. tuberculosis σB is essential for recognizing G−14 and G−13. (A) Alignment of amino acid sequences from region 2.1 to region 3.0 of principal and principal-like σ factors from M. tuberculosis (Mtb), M. smegmatis (Ms), and E. coli (Ec). Amino acids reported to be essential for recognizing the −10 element (8, 50) are indicated by black triangles, and those required to recognize the TGN element (14) are indicated by white triangles. (B) Activities of wild-type (WT) and mutated σB in recognizing rbpAp. **, P < 0.01; n.s., not significant. (C) Efficiencies of σB-E169K and σA-E374K in recognizing different promoters. *, P < 0.05; **, P < 0.01. (D) Position of E169 in mycobacterial σB modeled on the M. smegmatis σA-RNAP holoenzyme in complex with DNA (PDB accession no. 5TW1). Temp, template. (E) In vitro transcription from Rv1494p using RNAP containing M. tuberculosis wild-type σB or the E169K mutant (left) or wild-type σA or the E374K mutant (right). RbpA protein was added in all of these tests. (F) DNase I footprinting analysis of the Rv1494p complex with RNAP containing wild-type σB or the E169K mutant in the presence of RbpA.

To rule out the possibility that the low efficiency of σB-E169K in activating rbpAp was caused by poor expression of this protein, we compared the efficiencies of σB-E169K in initiating other promoters using an E. coli σB overexpression system. σB-E169K was defective in activating promoters containing either G−14 or G−13 but successfully activated promoters containing C−13 (Fig. 4C; also see Fig. S4). Consistent with previous observations for E. coli σS (24), which contains a K residue corresponding to E169 of M. tuberculosis σB (Fig. 4A), mutation or introduction of C−13 in promoters changed the efficiencies of transcription initiated by σB-E169K (Fig. S4). These data suggest that σB-E169K was well expressed in our system.

In the σA-RNAP-promoter structure from M. smegmatis (PDB accession no. 5TW1) (23), the E residue corresponding to E169 of σB is close to the template strand of the promoter around the −14 and −13 positions (Fig. 4D), suggesting that this residue may be directly involved in recognizing G−14 and G−13 in promoters. The structures of the σA-RNAP-promoter complex from Thermus aquaticus (PDB accession no. 4XLN) (10) (Fig. S3B) and the σ70-RNAP-promoter complex from E. coli (PDB accession no. 4YLN) (9) (Fig. S3C) also support this hypothesis. Moreover, σB-E169K was defective in initiating promoters containing either G−14 or G−13, when assessed using in vitro transcription assays (Fig. 4E), and could not effectively bind to a G−14G−13-containing promoter, compared with wild-type σB, in the DNase footprinting assay (Fig. 4F). These data indicate that the E residue in region 3.0 plays a key role in recognizing G−14 and G−13 in promoters. A sequence alignment showed that E169 of M. tuberculosis σB is conserved in mycobacterial σA and σB and E. coli σ70 (Fig. 4A), suggesting that recognition of G−14 and G−13 might also be applicable to other σ factors.

Other σ factors containing corresponding E169 also recognize G−14 and G−13.

To investigate whether G−14 and G−13 in promoters could be recognized by σ factors with a conserved E169 residue, we first used mycobacterial σ-overexpressing E. coli (20) and the σA-RNAP-RbpA transcription model from M. tuberculosis (21, 25) to compare the in vivo and in vitro efficiencies of mycobacterial σA in initiating promoters containing G−14 and/or G−13. Results similar to those observed for σB were obtained in both systems (Fig. 4C and E; also see Fig. S4).

In E. coli, a recent study identified nearly 15,000 TSSs (26). Promoters located upstream of these TSSs were also analyzed here. Among them, 8,091 contained the −10 element in the TANNNT sequence and over 64% of those promoters contained at least one G at the −14 and −13 positions upstream of the −10 element (Fig. 5A), suggesting that the prevalence of upstream G in E. coli promoters is similar to that in mycobacteria. Six promoters in E. coli (referred to as Ecp1 through Ecp6) (Fig. 5B) were chosen for a mutagenesis analysis. The mutation of either G−14 or G−13 decreased the promoter activities, and the introduction of either G−14 or G−13 led to increases in the promoter activities (Fig. 5C to E). These data suggest that activation by G−14 or G−13 for transcription initiation might be also used by E. coli.

FIG 5.

FIG 5

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Roles of G−14 and G−13 in activating E. coli promoters. (A) Percentages of promoters containing G−14 and/or G−13 upstream of TANNNT in the E. coli promoters. Promoter sequences were analyzed according to the TSSs characterized by Thomason et al. (26). (B) DNA sequences of Ecp1 to Ecp6 promoters. Nucleotides at the −14 and −13 positions are shown with a green background. (C to E) Effects of mutating G−14 and/or G−13 on the activities of Ecp1 to Ecp6. *, P < 0.05; **, P < 0.01; n.s., not significant. (F) Sequences of mutated Ecp1 promoters. Mutated nucleotides are shown in red. (G) Activities of mutated Ecp1 promoters.

To test whether the promoter containing only the −10 element and upstream G would also be functional in E. coli, we mutated the −35 element of the Ecp1 promoter (Fig. 5F) and found that this mutated promoter was still active, although the activity was obviously lower than the parental activity (Fig. 5G). Mutation of G−14G−13 to A−14A−13 in the −35-element-mutated Ecp1 dramatically decreased the promoter activity (Fig. 5G). These data suggest that a mechanism for recognizing promoters containing only a −10 consensus and upstream G might be employed by E. coli.

Although we have tested only the recognition of promoters containing −10 and upstream G in mycobacteria and E. coli, we aligned the amino acid sequences of σ factors from different bacteria. Region 2 and the E residue corresponding to E169 of M. tuberculosis σB are highly conserved in principal and principal-like σ factors from mycobacterial species and in principal σ factors from other bacteria (Fig. S5). These analyses suggest that recognizing promoters containing the −10 element and upstream G−14 or G−13 might be conserved in bacteria.

Promoters using G−14 or G−13 together with the −10 element in mycobacteria are predicted.

Because the activities of the mycobacterial promoters containing the −10 element and the upstream G were not dependent on the −35 consensus, we could simply use DNA sequences GHTANNNT, HGTANNNT, or GGTANNNT (with H representing A, T, or C) to predict promoters containing the conserved −10 element and the upstream G. In this way, thousands of putative promoters were identified in different mycobacterial species (Table 1). To test whether the promoter prediction was reliable, we examined the activities of several newly predicted promoters from M. tuberculosis and found that all tested promoters were functional and that their activities were dependent on the upstream G (Fig. S2D to F).

TABLE 1.

Predicted numbers of promoters containing the conserved −10 element and an upstream G in mycobacterial species

Species No. of promotersa
G−14H−13 G−14G−13 H−14G−13 Total
Mycobacterium tuberculosis 4,305 1,595 3,121 9,021
Mycobacterium bovis 4,334 1,587 3,116 9,037
Mycobacterium avium 2,170 769 1,895 4,384
Mycobacterium marinum 4,033 1,326 3,119 8,478
Mycobacterium smegmatis 2,443 1,036 2,779 6,258
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a

H represents A, C, or T.

DISCUSSION

Promoter recognition determines the specificity of transcription. Although three key promoter elements, i.e., −35, −10, and TGN, have been characterized in bacterial promoters for σ binding (4, 5), a large proportion of the promoters in M. tuberculosis contain only a conserved −10 element (18). How these promoters promote transcription initiation has not been well understood. In this study, we discovered a new type of promoter in mycobacteria that contains the −10 element and upstream G. This upstream G is located at −14 or −13 or both positions and, together with the −10 element, efficiently activates transcription initiation. Although bacteria may employ the −10 element together with nonconserved −35 (6) or transcriptional regulator binding regions (5, 27) or an A+T-rich UP element recognized by the α subunit of RNAP (28–30) to initiate transcription in some promoters, more than 70% of the identified promoters in M. tuberculosis contain G−14 and/or G−13, which suggests that recognition of promoters containing the −10 element and the upstream G would be a major mechanism for transcription initiation in this bacterium.

The mechanisms for the roles of G−14 and G−13 in transcription initiation remain largely unclear. Loss of protection of the DNA region from −45 to +25, a feature of the promoter open complex (22), was observed when the upstream G was mutated in DNase I footprinting analysis (Fig. 3C), suggesting that G−14 and G−13 function in promoter recognition and open complex formation. Since transcription initiation from promoters containing the −10 element and G−14 or G−13 does not require other promoter elements, the contributions of G−14 and G−13 to open complex formation should be essential. Details of the actions of G−14 and G−13 need to be elucidated. In addition to the σ factor, mycobacterial RNAP requires two additional proteins, RbpA and CarD, for transcription initiation (21, 25, 31, 32). These two proteins are both reported to bind DNA (33, 34) and are located close to the nucleotides at the −14 and −13 positions in the RNAP-promoter complex (23, 35, 36). Whether these two proteins are connected to the functions of G−14 and G−13 in mycobacteria is worthy of further study.

A previous study demonstrated that nucleotides at the −14 and −13 positions are important for promoter recognition by the principal-like σS in E. coli (24). The wild-type σS contains a K residue corresponding to E169 of σB and has a preference for C−13 in promoters, whereas the σS-K173E variant has an altered preference for G−13 (24). Structural mapping showed that the E residue in σ region 3.0 corresponding to E169 in σB is in close proximity to the template strand of promoter DNA with G−14 and G−13 (see Fig. S3 in the supplemental material) (9, 32). The template strand sequences for G−14 and G−13 are C−14 and C−13, and the E residue in σ has been reported to prefer a cytosine determinant (14, 37). These analyses support our hypothesis that the E169 residue in mycobacterial σB region 3.0 may directly recognize G−14 and G−13. Comprehensive mutagenesis in region 3.0 of σ70 in E. coli also indicated that E458 (corresponding to E169 in σB) is essential for interacting with the G−14-C−14 base pair in the TGN element (14, 34). These findings suggest that the amino acids in σ region 3.0 have a discriminatory role with respect to the −13 and −14 promoter positions. Previous studies using aptamer single-stranded DNA (ssDNA) found that free σ factor can bind to nontemplate-strand TG−13TAGAAT or G−13TA(C/T)AATGGGA (the underlined sequences are −10 elements), and mutational analyses showed that nucleotides at the −14 and −13 positions on the nontemplate strand are important for ssDNA binding (38, 39), suggesting that other possible mechanisms for recognizing the nucleotides on the nontemplate strand at the −14 and −13 positions may also exist.

Promoters containing the −10 element and upstream G would be a conserved mechanism for transcription initiation. The E residue in region 3.0 that is essential for recognizing this upstream G and the region 2 that is essential for recognizing the −10 element (6, 7) are highly conserved in principal and principal-like σ factors in mycobacteria, as well as in principal σ factors from other bacteria (Fig. S5), suggesting that activation of the upstream G in transcription may be applied by other bacteria, in addition to mycobacteria. Structures of RNAPs from T. aquaticus and E. coli in complex with promoter DNA support this hypothesis (9, 10). Furthermore, bioinformatic analyses of promoter sequences from E. coli, Salmonella enterica, and Corynebacterium glutamicum showed that G−14 and G−13 present higher frequencies than other nucleotides upstream of the −10 element (28, 40, 41). In this study, we have detected transcriptional activation by G−14 and G−13 in E. coli (Fig. 5). Situations in other bacteria need to be studied further.

Because the −35 region is not well conserved and the spacer between −10 and −35 varies in promoters, predicting bacterial promoters using the −10 and −35 elements continues to be a challenging approach (42, 43). Since the activities of promoters containing G−14 or G−13 do not require the presence of the −35 consensus in mycobacteria, we are able to predict promoters by adopting the GTANNNT or GNTANNNT sequences. Thousands of promoters in mycobacterial species were predicted in this way, and the activities of representative promoters were confirmed. Interestingly, promoters are located in both the intergenic region upstream of the genes and the genes themselves (data not shown). A recent study that characterized TSSs from E. coli showed that approximately two-thirds of the TSSs are located in genes and approximately one-half of them are oriented in the same direction as the genes (26), suggesting that, in addition to mRNA, antisense RNA, and small noncoding RNA, there may be other forms of transcripts with unknown functions. Systematic characterization of the bacterial promoters will enable exploration of the roles of these transcripts.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The bacterial strains used in this study are listed in Table S1 in the supplemental material. E. coli strains were routinely grown in Luria-Bertani (LB) broth or agar, at 30°C for the ΔlacZ strain or at 37°C for the DH5α and BL21(DE3) strains. M. smegmatis mc2155 was cultured at 37°C in 7H9 liquid medium (Difco) supplemented with 0.2% glucose, 25 mM NaCl, 0.2% glycerol, and 0.05% Tween 80 or on 7H10 agar plates (Difco) supplemented with 0.5% glycerol. Antibiotics were added when required.

Plasmid construction.

The plasmids and oligonucleotides used in this study are listed in Tables S1 and S2, respectively. All DNA fragments (including promoter fragments and genes) from E. coli were amplified using genomic DNA from the K-12 MG1655 strain as the template. All DNA fragments from M. tuberculosis were amplified from the H37Rv genomic DNA (product no. NR-14865; BEI Resources). PCR fragments were inserted into a linearized vector using the ClonExpress II one-step cloning kit (Vazyme, China). Mutations in the genes or promoters were introduced using the QuikChange II XL site-directed mutagenesis kit (Stratagene).

Protein purification.

The plasmid pMR4, expressing the RNAP core enzyme from M. tuberculosis with a His tag at the C terminus of the β′ subunit, was transformed into E. coli BL21(DE3). Protein expression was induced by the addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of ∼0.6 for the culture and another incubation at 24°C for 20 h. Cell pellets were collected and lysed by ultrasonication. RNAP was purified with Ni-resin, heparin column, and gel filtration chromatography as described previously, with modifications (21). A Superdex 200 column (GE Healthcare) was used in the gel filtration analyses in this study. The σ factors were expressed in fusion with His-SUMO-tev (mycobacterial σB) or His-tev (RbpA and mycobacterial σA with truncation of 165 amino acids at the N terminus) and purified by Ni-resin chromatography as described for the RNAP core enzyme. His-TEV protease expressed by the pRK793 plasmid was used to remove the tags of target proteins linked at the TEV protease site (for mycobacterial σA, σB, and RbpA), as described previously (44).

Promoter activity analysis in E. coli.

Promoters from E. coli or mycobacteria were fused to the promoterless lacZ gene in the pZT100 plasmid (45) and transformed into the E. coli K-12 ΔlacZ strain (20). Constructs of the mycobacterial promoters were cotransformed with a mycobacterial σ overexpression plasmid (pOVR-σA or pOVR-σB) (20). Plasmid pOVR200 (45) was cotransformed with the mycobacterial promoter constructs and used as the control. The β-galactosidase activities were tested in E. coli when the OD600 of the cultures reached ∼0.8, as described previously (46). The mean and standard deviation (SD) of three colonies tested in duplicate are shown, and Student's t test was used to compare the data between two groups.

Promoter activity analysis in mycobacteria.

Promoters from M. tuberculosis were fused to the promoterless egfp gene in the integrative plasmid pMV306 (47) and transformed into M. smegmatis. When the OD600 of each culture reached ∼0.8, cells were washed and resuspended in phosphate-buffered saline with 0.05% Tween 80 (PBST). The fluorescence intensities were tested in black 96-well plates. The promoter activities are shown as relative fluorescence units (RFU), determined as the fluorescence intensities per OD600. Two colonies in each group were tested in duplicate. Student's t test was used to compare the data between two groups.

In vitro transcription.

Multiple-round transcription reactions were performed as described previously, with minor modifications (21). Briefly, transcription was performed in 5 μl of transcription buffer (TB) (20 mM Tris-HCl [pH 7.9], 50 mM NaCl, 5 mM MgSO4, 1 mM dithiothreitol [DTT], 0.1 mM EDTA, and 5% glycerol). The RNAP holoenzyme was assembled by mixing the σ factor (400 nM σB or σA) and RNAP core (200 nM) for 10 min at 37°C. Where indicated, the RbpA protein was added to a final concentration of 800 nM. A promoter fragment (30 nM) amplified from the pZT100 construct was then added and incubated at 37°C for 10 min. Transcription was initiated by the addition of 50 μM limited nucleoside triphosphates (GTP and UTP for Rv1494p or GTP and ATP for Rv0005p) together with 1 μCi [α-32P]CTP. The reactions were carried out at 37°C for 20 min and were stopped by the addition of 1 volume of stop buffer (95% formamide, 0.025% bromophenol blue, 5 mM EDTA, 0.025% SDS, and 4 M urea). RNA was analyzed on a 20% denaturing PAGE gel (7 M urea), and ImageJ software was used to quantify the products in each gel.

DNase I footprinting.

DNase I footprinting analyses were based on a fluorescence labeling procedure described previously, with modifications (21). Briefly, 5′-6-carboxyfluorescein (FAM)-labeled promoters (40 nM) were mixed with 400 nM RNAP to form a complex in 10 μl TB. After incubation at 37°C for 20 min, samples were treated with 2 U/ml DNase I (Promega) for 1 min at 24°C. The reactions were stopped by the addition of 10 mM EDTA (pH 8.0). Samples were then loaded into an Applied Biosystems 3730xl DNA analyzer, and capillary electrophoresis results were analyzed with Sequencing Analysis software, version 5.2 (Applied Biosystems).

Promoter sequence analysis and promoter prediction.

Promoter sequences were analyzed on the basis of the knowledge that the −10 element was located 5 to 7 bp upstream of the identified TSSs. DNA sequences around the identified TSSs (from −49 to +1) in M. tuberculosis (19) were analyzed. The TANNNT sequence was used to map the potential −10 elements. The first T in this sequence was numbered as −12 and centered for analysis of the nucleotides at positions −15 to −13. Virtual Footprint (http://www.prodoric.de/vfp/index2.php) (48) was used to predict promoters in bacteria.

Supplementary Material

Supplemental material
supp_199_21_e00385-17__index.html (1.2KB, html)

ACKNOWLEDGMENTS

We thank Konstantin Brodolin for critically reading the manuscript, Heran Darwin for providing the pMV306 plasmid, and Jiaoyu Deng for providing the egfp fragment. Support from the 135 Research Project and the Core Facility and technical support from the Wuhan Institute of Virology are also acknowledged.

This work was supported by the National Natural Science Foundation of China (grant 31670134). Y.H. was supported by the Youth Innovation Promotion Association CAS (grant 2013220).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00385-17.

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