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Open Access

Peer-reviewed

Research Article

An antisense RNA regulates production of DnaA and affects sporulation in Bacillus subtilis

  • Emma L. Sedivy,

    Roles Conceptualization, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America

    ⨯
  • Janet L. Smith,

    Roles Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – review & editing

    Affiliation Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America

    ⨯
  • Alan D. Grossman

    Roles Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing

    adg@mit.edu

    Affiliation Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America

    ⨯

Abstract

DnaA is the replication initiator and a transcription factor in virtually all bacteria. Although the synthesis and activity of DnaA are highly regulated, the mechanisms of regulation vary between organisms. We found that production of DnaA in Bacillus subtilis is regulated by an antisense RNA that overlaps with the 5’ untranslated region upstream of the dnaA open reading frame. We initially observed this RNA in in vitro transcription experiments and found that its production was inhibited by DnaA. This RNA, now called ArrA for antisense RNA repressor of dnaA, is made in vivo. We identified the arrA promoter and made a mutation that greatly reduced (or eliminated) production of ArrA RNA in vitro and in vivo. In vivo, this arrA promoter mutation caused an increase in the amount of mRNA and protein from dnaA and dnaN, indicating that arrA expression normally inhibits expression of the dnaA-dnaN operon. The arrA mutation also caused a delay in sporulation that was alleviated by loss of sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in some members of the Bacillus genus, indicating that arrA has evolved in at least some endospore-forming bacteria to modulate production of DnaA and enable timely and robust sporulation.

Author summary

DnaA is the highly conserved replication initiator and transcription factor found in virtually all bacteria. The synthesis and activity of DnaA are highly regulated, and different types of bacteria use different mechanisms to control this key protein. We found that DnaA production in Bacillus subtilis is inhibited by an antisense RNA that overlaps with the 5’ untranslated region of the dnaA mRNA. In the absence of this antisense RNA, there was an increase in the amount of dnaA mRNA and protein. There was also a delay in sporulation that depends on sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in several members of the Bacillus genus, indicating that it has evolved to temper production DnaA and enable robust sporulation in these species.

Citation: Sedivy EL, Smith JL, Grossman AD (2025) An antisense RNA regulates production of DnaA and affects sporulation in Bacillus subtilis. PLoS Genet 21(5): e1011625. https://doi.org/10.1371/journal.pgen.1011625

Editor: Kai Papenfort, Friedrich-Schiller-Universitat Jena, GERMANY

Received: February 17, 2025; Accepted: April 25, 2025; Published: May 14, 2025

Copyright: © 2025 Sedivy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: Research reported here is based upon work supported, in part, by a NSF GRFP awarded to ELS and by the National Institute of General Medical Sciences of the National Institutes of Health under award number GM41934, GM122538, and GM148343 to ADG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

DnaA is required for the initiation of DNA replication in virtually all bacteria, and also functions as a transcription factor {reviewed in [1,2]}. DnaA binds to sequences in the origin of replication (oriC) and promotes DNA unwinding and subsequent assembly of the replisome {reviewed in [3–5]}. The activity of DnaA in replication initiation and at oriC is highly regulated, although many of the mechanisms of regulation vary between different bacterial species [6,7].

In most species, including B. subtilis and E. coli, dnaA is immediately upstream of and co-transcribed with dnaN (encoding the sliding clamp, a.k.a., the β-clamp, of DNA polymerase) (Fig 1A). DnaA represses transcription of the dnaA-dnaN operon, creating a homeostatic feedback loop that is one of the more highly conserved mechanisms in bacteria to regulate expression of dnaA {[2] and references therein}.

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Fig 1. Map of the oriC region of the chromosome and effects of arrA on DnaA and sporulation.

A. Map of the oriC region of the B. subtilis chromosome. Distances are shown approximately to scale. The black horizontal line represents the chromosome and rpmH, dnaA, and dnaN are indicated as white rectangles with arrows indicating the direction of transcription. Transcription start sites and terminators are indicated by arrows and stem-loops (⚲), respectively, above and below the chromosome. The red arrows indicate the transcription start site of arrA. The transcription terminator between dnaA and dnaN allows partial readthrough, as represented by a smaller stem-loop (⚲) symbol. Inset: The DnaA binding sites upstream of dnaA are indicated with small triangles showing location and direction. Black triangles indicate consensus DnaA binding sites; lighter shaded triangles are proportionally less similar to the consensus DnaA binding motif [13]. B. Pathway connecting arrA to Spo0A and the initiation of sporulation. The transcription factor Spo0A serves as the master regulator for entry into sporulation. Spo0A is activated by phosphorylation via a phosphorelay that involves transfer of phosphate from histidine protein kinases (primarily KinA and KinB, although others can also contribute) to Spo0F, then from Spo0F to Spo0B (not indicated), and finally to Spo0A. Spo0A~P is essential for the initiation of sporulation and activates several genes including the sporulation genes spoIIA, spoIIE, and spoIIG. DnaA directly activates transcription of sda, whose product inhibits the kinases (KinA and KinB) needed for the activation of Spo0A and the initiation of sporulation. We found that transcription of arrA serves to decrease expression of dnaA, and that this function of arrA is crucial for the proper timing of sporulation.

https://doi.org/10.1371/journal.pgen.1011625.g001

DnaA also binds sequences in other promoter regions and in many cases directly modulates transcription of the associated genes {reviewed in [2]}. In B. subtilis, eight promoter regions are strongly bound by DnaA [8–14] and there are many transcription units whose expression is affected both directly and indirectly. By virtue of its effects on gene expression, DnaA regulates sporulation, competence development, and the response to DNA damage [8,12,15,16].

DnaA can inhibit the initiation of sporulation by activating transcription of the sporulation inhibitory gene sda [9,11,15]. The sda gene product inhibits protein kinases needed for the initiation of sporulation [15,17–19] (Fig 1B). In this way, when there is an increase in the amount of active DnaA (e.g., when there is replication arrest), there is a subsequent increase in transcription of sda and sporulation initiation is inhibited [12,15,17,20].

Here we report that a small RNA (sRNA) encoded antisense to the 5’ end of the dnaA operon represses dnaA and dnaN in B. subtilis. This RNA, named ArrA for anti-sense RNA repressor of dnaA, overlaps with the 253 bp 5’ untranslated leader region of the dnaA open reading frame and was previously identified in transcriptome analyses and called S2 [21]. We found that an arrA promoter mutant that had reduced or eliminated production of ArrA RNA caused an increase in DnaA and DnaN mRNA and protein and a delay in sporulation initiation. We also found that the delay in sporulation was alleviated by loss of sda, indicating that the effects of arrA on sporulation were mediated by its effect on DnaA and subsequent effect on expression of sda (Fig 1B). Together, our results indicate that the normal function of arrA is to reduce the amount of DnaA and DnaN in the cell and help enable proper initiation of sporulation, and that this antisense control of dnaA by arrA is critical for normal cellular physiology. Lastly, although there are not many analyses of the small RNAs in various Bacilli, ArrA appears to be conserved in at least some members of the Bacillus genus.

Results

In vitro transcripts from the promoter region of the dnaA operon

The promoter region of the dnaA-dnaN operon is part of oriC and contains several DnaA binding sites (Fig 1A). When DnaA is bound to some of these sites, transcription of the operon is decreased {reviewed in [1,2]}. In the course of analyzing transcription from the dnaA promoter (PdnaA) in vitro and the activity of DnaA as a transcriptional repressor, we observed a transcript that was more abundant than that initiating from the dnaA promoter (Fig 2). The in vitro transcription reactions contained RNA polymerase that had been purified from B. subtilis, the major sigma factor SigA, ribonucleotides, appropriate buffer and 32P-UTP for visualization of the transcripts. We used a linear DNA template (a 1,431 bp PCR product) that was expected to give a run-off transcript of ~250 nucleotides from PdnaA (Fig 2A).

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Fig 2. In vitro transcription from the dnaA promoter and the antisense arrA promoter.

A. Cartoon of the template and transcripts for in vitro transcription. The linear DNA template used for run-off in vitro transcription was a 1,431 linear PCR product amplified from a plasmid containing the B. subtilis dnaA promoter region (enlarged in Fig 1A). This template fragment contains plasmid sequences (gray line) and sequences from the dnaA region (black line). Triangles indicate DnaA binding sites as described in Fig 1A. The 252-nucleotide transcript expected from PdnaA is depicted as a dotted black line above. The transcript observed that led to the identification of arrA is depicted as a dotted line below and labeled in red as antisense transcript. B. In vitro transcription reactions and repression of PdnaA and ParrA by DnaA. In vitro transcription reactions contained 3.5 nM of the DNA template (Fig 2A), 50 nM RNA polymerase and 300 nM sigma-A. Lane 1: Markers, with sizes indicated to the left. Lane 2: Products of in vitro transcription in the absence of DnaA. Products from PdnaA and ParrA are marked with red asterisks and indicated to the right of the figure. Lane 3: Products of in vitro transcription in the presence of DnaA-ATP (250 nM).

https://doi.org/10.1371/journal.pgen.1011625.g002

As expected, the in vitro transcription reactions produced a product that was ~ 250 nucleotides (Fig 2B, lane 2), corresponding to the predicted product from PdnaA. However, we also observed two transcripts (labeled arrA) that were >1,000 nucleotides and apparently more abundant than that from PdnaA. Based on size, these larger transcripts might initiate from far upstream of PdnaA and be in the same orientation, or initiate downstream of PdnaA and go in the opposite orientation.

We found that production of the larger transcripts was repressed by DnaA. Addition of DnaA to the in vitro transcription reactions caused a large decrease in the amount of the larger transcripts, and also appeared to modestly repress the transcript from PdnaA (Fig 2B lane 3). Repression of both of the larger transcripts by DnaA indicated that they likely initiate from a promoter that is close to DnaA binding sites, making it likely that the promoter was downstream from and in the opposite orientation of PdnaA (Fig 2A). We do not know why there are two bands, but they could be isoforms of the same transcript, they could initiate at the same promoter but end at different places, or one could result from end-to-end transcription. Experiments described below allowed us to focus on a single promoter driving a transcript that is anti-sense to the dnaA mRNA. Based on results described below, we named the antisense gene arrA (pronounced R-A) for antisense RNA repressor of dnaA.

The arrA promoter

We scanned the DNA sequence downstream from and in the opposite orientation of PdnaA for possible promoters that could generate the observed transcript in vitro and be repressed by DnaA. We found a good candidate for a promoter that would likely be recognized by the major form of RNA polymerase (containing SigA). There is a perfect match (5’-TATAAT) to the consensus −10 region and a reasonable match (5’-TacACA) to the consensus −35 region (consensus: TTGACA) with 16 bp separating them (Fig 3A). Further, there are two DnaA binding sites that overlap this promoter, one in −35 region and another between the −10 and −35 regions (Fig 3A), and additional DnaA binding sites downstream from this putative promoter (Figs 2A and 3B).

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Fig 3. In vitro transcription from the arrA promoter.

A. Sequence of the arrA promoter region. The putative −10 and −35 regions of the arrA promoter are indicated and shown in bold capitals on the bottom strand going leftward. The −10 sequence is a perfect match to the consensus, and the −35 sequence (TACACA) has two mismatches from the consensus (TTGACA). DnaA binding sites are indicated above the top strand and their sequences shown in italic capital letters. The arrA1 allele (shown in red) changes the −10 (TATAAT) to GGGGGG. B. Cartoon of template and transcripts for in vitro transcription. The template used for in vitro transcription was a 948 linear PCR product amplified from a plasmid containing the B. subtilis dnaA and arrA promoter regions. This plasmid was engineered to include transcription terminators (⚲): One from B. subtilis serO/aroC (‡) downstream from the dnaA promoter and another from gatB/yerO (†) downstream from the arrA promoter. The portion of the template derived from the dnaA/arrA region is shown in black, and the regions derived from the plasmid and the transcription terminators are shown in grey. Other features are as described in Fig 1A. C. In vitro transcription with wild type and arrA1 mutant templates. In vitro transcription reactions contained 2 nM of the DNA template (Fig 3B), 30 nM RNA polymerase and 45 nM sigma-A. Lane 1: Markers, with sizes indicted on the left. Lane 2: Products of in vitro transcription using a template with wild type promoters; transcripts from PdnaA and ParrA are indicated to the right of the figure. Lane 3: Product of in vitro transcription using a template with the arrA1 mutant promoter (and wild type dnaA promoter).

https://doi.org/10.1371/journal.pgen.1011625.g003

We found that the putative promoter was responsible for the arrA transcript. We altered the −10 region, changing the sequence from 5’-TATAAT to 5’-GGGGGG {arrA1} and then used a template containing this mutant promoter in in vitro transcription reactions. In these experiments, we used a 948 bp template that contains both PdnaA and ParrA, along with transcription terminators that we introduced (Fig 3B). As expected, there were transcripts of ~300 and ~500 nucleotides corresponding to those predicted from PdnaA and ParrA, respectively (Fig 3C, lane 2). We found that the arrA1 mutation abolished the arrA transcript but had little or no detectable effect on transcription from PdnaA (Fig 3C, lane 3). Together, these results indicate that, at least in vitro, there is an antisense RNA that is complimentary to the 5’ untranslated region of the dnaA transcript and that this transcript initiates at ParrA and is repressed by DnaA. These results also indicate that arrA is unlikely to be directly inhibiting transcription from the dnaA promoter as the amount of the dnaA transcript did not detectably increase with the arrA1 mutant template.

arrA RNA is made in vivo

The arrA transcript has been observed in whole-genome transcriptome studies of B. subtilis and was annotated as transcript S2 [21]. There is very little ribosome footprint density in this region [22], indicating that the RNA is likely non-coding. In contrast to our observations from in vitro transcription where the arrA transcript was more abundant than that from the dnaA promoter, there is approximately 20-fold more dnaA mRNA than arrA RNA during exponential growth in LB and minimal media [21,22]. We suspect that the relatively low level of arrA RNA in vivo compared to in vitro is likely due to repression of transcription by DnaA in vivo, similar to the relative repression of the two promoters that we observed in vitro (Fig 2B).

We found that production of the arrA transcript in vivo was dependent on the sequences we identified as the putative arrA promoter. We introduced the arrA1 allele into its normal chromosomal location in the dnaA-dnaN operon, extracted RNA from cells growing exponentially in LB medium and used northern blotting to detect the arrA RNA. Transcripts were probed with a radiolabeled oligonucleotide (Fig 4A, indicated as ssDNA oligo) complementary to the 5’ portion of the arrA transcript.

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Fig 4. Mutations in the arrA promoter region abolish transcription in vivo.

A. Map of the dnaA promoter region with probe and transcripts indicated. rpmH and dnaA are shown in white (not to scale). The radiolabeled oligonucleotide that was used in northern blotting hybridizes near the 5’ end of the arrA transcript and is indicated in red (ssDNA oligo). A cartoon of the observed transcripts is shown below. The 667 nucleotide transcript extends through rpmH and the 270-nucleotide transcript is consistent with either a weak transcriptional terminator or RNA processing of the longer transcript. Promoters and DnaA binding sites are indicated as in figures above. B. Northern blot probing for arrA RNA. RNA from cells in mid-exponential growth in LB was probed using the radiolabeled ssDNA oligonucleotide that hybridizes near the 5’ end of the arrA transcript. Lane 1: RNA from wild type cells. Lane 2: RNA from arrA1 mutant cells. The small and large bands correspond to the 270 and 667 nucleotide transcripts drawn in panel A. C. The same gel in B was stained with SYBR Gold prior to transfer to visualize total RNA. Lane 3 contains RNA markers (sizes in nucleotides on the right). Images of the blot and stained gel are shown in S1A Image in S1 File.

https://doi.org/10.1371/journal.pgen.1011625.g004

We observed an approximately 270 nucleotide transcript (Fig 4B lane 1) that hybridized to the arrA probe, consistent with transcription initiating at ParrA and terminating in the intergenic region between dnaA and rpmH (Fig 4A). This is consistent with the 267 nucleotide transcript observed in analyses of Rend-seq data [23]. We also observed a ~ 670 nucleotide transcript (Fig 4B) consistent with transcription initiating at ParrA and extending through rpmH and terminating at the rpmH terminator (Fig 4A). Notably, both of these transcripts were eliminated in the arrA1 mutant. That is, there was no detectable RNA corresponding to the two transcripts in this mutant (Fig 4B, lane 2). There were similar amounts of total RNA in each of the two samples (lanes 1, 2) as determined by staining the gel with SYBR Gold prior to transfer (Fig 4C, lanes 1, 2). We conclude that the arrA1 mutation reduced transcription of arrA to levels below our limit of detection, both in vivo and in vitro, and that the residues changed are most likely in the −10 region of the arrA promoter.

Effects of arrA on dnaA and dnaN

We found that arrA was important in vivo for proper control of dnaA and dnaN. We measured the amount of dnaA and dnaN mRNA in arrA+ (wild type) and arrA1 mutant cells during exponential growth in defined minimal medium using RT-qPCR with primers internal to and near the 3’ end of each open reading frame (S1 Table). We found that cells with the arrA1 mutation had approximately twofold more mRNA for both dnaA and dnaN than wild type cells (Fig 5A). These results indicate that arrA normally functions to reduce mRNA levels of dnaA and dnaN.

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Fig 5. arrA affects expression of dnaA, dnaN, and ywlC.

Expression of dnaA and dnaN (mRNA and protein) ywlC-lacZ expression were measured in wild type (filled circles) and arrA1 (open circles) mutant cells during exponential growth in defined minimal medium with glucose. A. The arrA1 mutant has elevated levels of dnaA and dnaN mRNA. The amount of dnaA and dnaN mRNA was measured using RT-qPCR. The amount of RNA from each gene was normalized to that from gyrA. Error bars show the mean and standard error of the mean from three independent experiments. mRNA from both dnaA and dnaN is significantly elevated in the arrA1 mutant (ELS393) compared to that in wild type (AG174) (P-values 0.0057 and 0.0021, respectively, in a two-tailed Student’s t-test). B. The arrA1 mutant has elevated levels of DnaA and DnaN protein. The amount of DnaA and DnaN protein was measured by western blotting. The amount of lysate loaded was normalized to the OD600 of the culture at the time of sampling. The hybridization membrane was cut between the expected molecular weights of DnaA and DnaN and quantitative western blotting was performed using antibodies against each protein. The data were normalized to the wild type samples for each protein. The amount of both DnaA and DnaN is significantly elevated in arrA1 cells (ELS393) (four biological replicates) compared to that in wild type (AG174) (P-values of 0.044 and 0.018, respectively, in a two-tailed Student’s t-test). An image of the blot is shown in S1B Image in S1 File. C. Transcription of ywlC is reduced in the arrA1 mutant. β-galactosidase specific activity from PywlC-lacZ was determined at different times during exponential growth. Data are from two independent cultures for each strain and three or four time points for each culture. Error bars represent the standard error of the mean of all seven samples from each strain. Expression from PywlC was lower in the arrA1 mutant (grey) compared to wild type (black) (P-value 2.3 × 10−7 in a two-tailed Student’s t-test).

https://doi.org/10.1371/journal.pgen.1011625.g005

The arrA1 mutant also had increased amounts of DnaA and DnaN protein. We measured the amount of DnaA and DnaN in western blot experiments using antibodies specific to each protein. We found that there was approximately twofold more DnaA and DnaN in the arrA1 mutant than in wild type cells (Fig 5B), consistent with the observed change in the amount of mRNA.

We also found that the arrA1 mutant had changes in expression of ywlC, a gene that is repressed by DnaA [8,9,11,12,16]. We found that expression of a PywlC-lacZ fusion was reduced in the arrA1 mutant (Fig 5C), likely due to the increased amount of DnaA. Together, our results indicate that arrA normally reduces production of DnaA and DnaN by causing a decrease in the amount of dnaA and dnaN mRNA, and that this regulation is important for proper control of gene expression.

Little or no effect of arrA on growth rate and replication initiation

Growth.

The growth rate of the arrA1 mutant was virtually indistinguishable from that of isogenic arrA+ cells (Table 1). In rich medium (LB), the doubling times were 21.8 ± 1.3 and 22.3 ± 1.3 minutes for wild type and the arrA1 mutant, respectively. In sporulation medium (DSM), the doubling times were 36 ± 1 and 36.5 ± 0.6 minutes for wild type and arrA1, respectively (Fig 6A and Table 1). Similarly, in defined minimal medium with glucose as carbon source (S7), the doubling times were 64 ± 5 for both wild type and the arrA1 mutant (Table 1). Based on these results, we conclude that there is little to no detectable effect of arrA on growth rate under the conditions tested.

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Table 1. Growth of and replication in arrA1 mutants.

https://doi.org/10.1371/journal.pgen.1011625.t001

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Fig 6. Effects of arrA and sda on sporulation and early sporulation gene expression.

Sporulation and expression of an early sporulation gene (PspoIIA-lacZ) were measured in cells growing in the sporulation medium DSM. Time 0 represents entry into stationary phase. A, B. Growth (OD600; diamonds) and expression of PspoIIA-lacZ (specific activity; circles) in arrA+ (filled symbols) and arrA1 mutant cells (open symbols). A. Wild type sda (sda+) strains: arrA+ (wt; strain ELS488) and arrA1 (strain ELS490). One representative experiment of three independent experiments is shown. B. ∆sda strains: arrA+ (ELS522), and arrA1 (ELS526). One representative experiment of two independent experiments is shown. C. Percent sporulation (% spores per viable CFU) was determined 8 hrs (left) and 24 hrs (right) after entry into stationary phase. Strains used were: wt (AG174); arrA1 (ELS393) and ∆sda arrA+ (ELS484); ∆sda arrA1 (ELS501). The difference in sporulation between wild type (1.3%) and arrA1 (0.24%) (both sda+) at 8 hrs was statistically significant (P = 0.021 using ratio paired t-tests).

https://doi.org/10.1371/journal.pgen.1011625.g006

Replication.

Increases in the amount or activity of DnaA or DnaN in the cell can cause an increase in replication initiation [24,25], although it is not clear how large an increase is needed to cause a detectable effect in B. subtilis. Replication initiation in B. subtilis is also controlled by the negative regulator YabA [10,25–30], and the activity of YabA might compensate for modest increases in DnaA and DnaN levels.

We tested whether the loss of the arrA transcript and subsequent increase in DnaA and DnaN had an effect on replication initiation. We measured the ratio of the amount of DNA from the origin region (ori) relative to that of the terminus region (ter) by quantitative PCR (qPCR). The ratio of these two regions is a measure of replication and alterations in replication initiation can cause changes in the ori to ter ratio (ori/ter). The ori to ter ratio in cells growing exponentially in defined minimal medium was 1.28 ± 0.05 and 1.49 ± 0.11 for wild type and the arrA1 mutant, respectively, corresponding to a 1.16-fold increase in the arrA1 mutant (Table 1). Although this increase was consistently observed in our experiments, it was not statistically robust (p = 0.096; paired t-test). The apparent increase in ori/ter caused by arrA1 is less than the ~ 1.3-fold and ~1.4-fold increases caused by the hyperactive allele dnaA1 or a yabA null mutation, respectively, both of which cause a growth defect [31]. Replication overinitiation caused by loss of yabA also leads to increased sensitivity to genotoxic stress [32]. Because the effects of arrA1 on replication and growth are much less than those caused by loss of yabA, we have not pursued possible alterations in sensitivity to genotoxic stresses. Rather, we focused on the more noticeable and robust effects on gene expression and sporulation.

arrA is needed for normal sporulation initiation

Alterations in the amount of active DnaA [15,33,34] are known to inhibit sporulation initiation [15,34,35]. Because arrA affects DnaA, we measured its effects on sporulation initiation. We verified that the amount of DnaA was increased in the arrA1 mutant when cells were grown in sporulation medium. Similar to growth in minimal medium (Fig 5B), there was an increase in the amount of DnaA and DnaN in the arrA1 mutant during growth in DSM sporulation medium (S1 Fig). As expected, the increased amount of protein was maintained for at least several hours after entry into stationary phase (S1 Fig). Although SigA-mediated transcription decreases in stationary phase, the existing DnaA and DnaN protein would not be signficantly diluted because cells are no longer growing rapidly.

We measured expression of spoIIA, an operon essential for and expressed early during sporulation, using a fusion of the spoIIA promoter to lacZ (spoIIA-lacZ). We previously used spoIIA-lacZ fusions to monitor effects of DnaA and replication stress on sporulation [34–36]. Transcription of spoIIA is activated by the phosphorylated form of the transcription factor Spo0A {reviewed in [37,38]} and its expression is reduced under conditions of increased DnaA protein or activity [8,16,34].

As expected, in wild type cells transcription of spoIIA-lacZ increased shortly after entry into stationary phase under sporulation conditions (Fig 6A). In contrast, expression of spoIIA-lacZ was reduced in the arrA1 mutant (Fig 6A). These results indicate that arrA is important for normal expression of an operon that is essential for and activated early during sporulation.

We also found that production of spores was altered in the arrA1 mutant. We measured sporulation approximately eight hours after cells entered stationary phase (the start of sporulation). There was an approximately five-fold difference in sporulation between wild type (1.1 x 107 spores/ml; 1.3% sporulation) and the arrA1 mutant (2.3 x 106 spores/ml; 0.24% sporulation) (Fig 6C). By 24 hours after the initiation of sporulation, there was no significant difference in sporulation levels of wild type (6.5 x 107 spores/ml; 18% sporulation) and the arrA1 mutant (3.0 x 107 spores/ml; 12% sporulation) (Fig 6C). Based on these results, we conclude that the initiation of sporulation is delayed in the arrA1 mutant, but that the mutant seems to eventually achieve wild type levels of sporulation, and that the normal function of arrA is to enable proper and robust sporulation initiation.

The near normal level of sporulation that is eventually reached is most likely indicative of cells entering the sporulation pathway in a much more asynchronous manner than that caused by the initial transition to stationary phase. The delay in the arrA mutant is reminiscent of that in a kinA (spoIIJ) null mutant. kinA encodes a histidine protein kinase that is involved in donating phosphate to and activating the transcription factor Spo0A [39]. The phosphorylated form of Spo0A (Spo0A~P) directly activates transcription of spoIIA (and other genes) {reviewed in [37,38]}. A kinA null mutant has a decrease in expression of spoIIA and a delay in sporulation [39,40]. Based on the similar phenotypes, it seems that loss of arrA might cause a decrease in KinA activity.

arrA affects sporulation through the sporulation inhibitory gene sda

Several mutations and conditions that affect replication and cause an increase in the amount or activity of DnaA also inhibit the initiation of sporulation [8,15,16,33,34,36]. In these cases, DnaA-mediated inhibition of sporulation is due to increased expression of the sporulation-inhibitory gene sda. Transcription of sda is directly activated by DnaA [9,12,15] and sda encodes a small protein that binds to and inhibits histidine protein kinases (including KinA) that are needed for activation (phosphorylation) of the transcription factor Spo0A [15,17–19] (Fig 1B).

We found that the effect of arrA1 on sporulation was mediated by sda. In an sda null mutant, expression of spoIIA-lacZ was virtually indistinguishable between arrA+ and arrA1 mutant cells (Fig 6B). Additionally, the sporulation efficiencies of arrA+ and the arrA1 mutant were also very similar in the absence of sda at both 8 and 24 hours (Fig 6C). Together, our findings indicate that the normal function of arrA is to dampen expression of dnaA, which in turn leads to decreased expression of the sporulation inhibitor sda and enables proper sporulation initiation.

Possible conservation of arrA in some Bacillus species

We searched the literature for evidence of possible small antisense RNA in the dnaA promoter region in other organisms. Many transcriptome analyses have not captured small RNAs, but of those that did, ArrA-like RNAs were not detected in Escherichia coli, Vibrio natrigens, Caulobacter crescentus, Staphylococcus aureus, members of the Clostridia genus, several different strains of Streptococcus pneumoniae, or Enterococcus faecalis [22,41–51].

Antisense RNA in the dnaA region in B. licheniformis.

An antisense RNA in the dnaA region was identified in B. licheniformis in transcriptome analyses [52,53]. Additionally, we identified a potential promoter for the antisense RNA between dnaA and its promoter (Fig 7A). There is a possible −10 region (5’-TATAGT) that has one mismatch from consensus (5’-TATAAT) and a possible −35 region (5’-ATGAAA) that has two mismatches from consensus (5’-TTGACA). Furthermore, there are two DnaA binding sites flanking the putative −10 region that are identical to those in B. subtilis (Fig 7A). Overall there is high sequence similarity in this region between B. subtilis and B. licheniformis, including the spacing of the two DnaA binding sites, although the two predicted promoters are offset by 5 bp. Based on this information, we suspect that B. licheniformis contains arrA and it is regulated and functions similarly to that of B. subtilis.

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Fig 7. Identification of possible antisense promoters with DnaA binding sites in various Bacillus species.

Sequences (5’ to 3’) from downstream of the dnaA promoter and in the vicinity of the arrA promoter from B. subtilis were compared and aligned using the ClustalOmega algorithm in JalView. We aligned the sequences by the conserved DnaA binding site (at the left of each sequence shown). Sequences matching a DnaA binding site are in capital letters, italicized, and shaded grey. The sequences of putative promoters (−10 and −35) for an antisense RNA (arrA) are shown in capital letters, bold and underlined. The consensus promoter sequence is shown above. Note: these are drawn on the anti-sense strand to dnaA, so the sequences are complimentary to the traditional −10 (TATAAT) and −35 (TTGACA). A. Comparison of the regions from B. subtilis and B. licheniformis. The B. subtilis arrA transcription start site is in bold, with a bent arrow on top indicating the direction of transcription. B. Sequences from different species are divided into four groups based on similarities and differences to B. subtilis and B. licheniformis. Groups I, II, and III, have two or three DnaA binding sites and the spacing between them is conserved. They all have a putative promoter that is antisense to dnaA and in all cases, the spacing between the −10 and −35 is 16 bp. I. Regions from 14 Bacillus species that are most similar to that in B. subtilis. There are 16 bp between the putative −10 and the downstream DnaA binding site, and 2 bp between the −10 and the upstream DnaA binding site. II. There is one member of this group, and the sequences are very similar to Group I, but the putative promoter elements are shifted by 2 bp leaving 14 bp between −10 and the downstream DnaA binding site and 4 bp between the −10 and the upstream DnaA binding site. III. Regions from two species are most similar to that from B. licheniformis. There are 11 bp separating the putative −10 and the downstream DnaA binding site, and 7 bp separating the putative −10 and the upstream DnaA binding site. IV. The arrA promoter does not appear to be present in B. cereus and members of its phylogenetic group, including B. anthracis and B. thuringiensis.

https://doi.org/10.1371/journal.pgen.1011625.g007

Possible antisense promoter in the dnaA region of other Bacilli.

We analyzed the region between the promoter and dnaA of 36 different Bacillus species (S2 Table) for possible promoters and DnaA binding sites, similar to those found in B. subtilis and B. licheniformis (Fig 7A). We found sequences similar to the consensus promoter in 17 additional regions analyzed (of 36) and grouped these based on whether the spacing between the −10 and the downstream DnaA binding site was more like that of B. subtilis (16 bp) (Fig 7B, Groups I, II) or B. licheniformis (11 bp) (Fig 7B, Group III). Fourteen had 16 bp (Fig 7B, Group I) and one had 14 bp (Fig 7B, Group II) between the putative −10 and the downstream DnaA binding site identical to that in B. subtilis. Two organisms had spacing between the putative −10 and the downstream DnaA binding site that is identical to that in B. licheniformis (Fig 7B, Group III). In all cases, the distance between the upstream and downstream nucleotide of the two DnaA binding sites (one downstream of the −10 and the other between the −10 and −35) is 24 bp even though the exact spacing relative to the putative promoter is somewhat different. We did not find similar promoter-like sequences for a possible antisense RNA in the dnaA region of the Bacillus cereus group, including B. cereus, B. anthracis, and B. thuringiensis (Fig 7B, Group IV). Together, these analyses indicate that an arrA-like transcript is likely made in several, but not all, Bacillus species and that its expression is repressed by DnaA. We suspect that its function in the modulation of expression of the dnaA-dnaN operon is conserved.

Discussion

We identified arrA as an antisense regulator of the dnaA operon. Transcription of arrA is repressed by DnaA and loss of arrA transcription caused an increase in the amount of DnaA and DnaN and a decrease in sporulation initiation. Below, we discuss arrA in the context of mechanisms regulating dnaA, two sRNAs that have been identified that are antisense to dnaA, and the effects of arrA on sporulation and its conservation.

Strategies for regulating DnaA

Bacteria use many different mechanisms to regulate DnaA levels and activity. Some mechanisms are highly conserved, whereas others are specific to particular taxa. For example, autoregulation by DnaA to repress its own promoter is highly conserved [2]. Also, in all organisms, the ATP-bound form of DnaA is needed for cooperative and proper binding to oriC to enable replication initiation {reviewed in [3–5]. Although virtually all organisms regulate cooperative binding of DnaA to oriC, the mechanisms used to control binding can be quite different [6,7]. For example, E. coli and related species regulate ATP hydrolysis and exchange, have mechanisms to titrate DnaA from oriC, and can sequester oriC to inhibit access by DnaA. In contrast, B. subtilis and apparently many other Gram positive bacteria use anti-cooperativity proteins to modulate proper binding of DnaA to oriC. Additional mechanisms modulating DnaA include regulated proteolysis [54] and trans-translation [55].

DnaA and arrA

Our work shows that arrA is a negative regulator of DnaA. Loss of production of arrA causes an increase in the amounts of DnaA mRNA and protein. arrA does not appear to affect the activity of the dnaA promoter as loss of arrA transcription had no detectable effect on the dnaA promoter in vitro. Rather, the simplest model for arrA function is that the arrA transcript base pairs with the dnaAN transcript causing a decrease in stability of the dnaAN mRNA and a corresponding decrease in protein production. The in vitro results show that transcription of arrA is repressed by DnaA. This creates a positive feedback loop in which DnaA indirectly activates its own production by inhibiting an inhibitor. Thus, DnaA functions both as a repressor (direct) and activator (indirect, through arrA) of its own production.

sRNAs and dnaA in B. subtilis

Two small RNAs, S2 and S4, that are antisense to dnaA were identified in genomic analyses of sRNAs in B. subtilis [21,56]. S2 is 267 nucleotides and corresponds to arrA. S4 is ~ 770 nucleotides, initiates from within the dnaA open reading frame, overlaps and shares a terminator with arrA, increases dramatically by three hours into sporulation, and is most likely transcribed by RNA polymerase containing the sporulation-specific sigma factor SigE [21]. The global transcriptomic analyses indicated that, like S4, S2 appeared to increase during sporulation. This apparent increase was postulated to be from a potential promoter recognized by RNA polymerase containing the sporulation-specific SigK [56]. However, we believe that the overlap between S4 and S2 and the sporulation-specific increase in S4 led to the proposal that S2 is expressed during sporulation [21]. Although the function of S4 RNA and any significance in its overlap with and possible sporulation-specific expression of S2 are not known, it is clear from our results that S2 (arrA) is expressed and functional during growth and the initiation of sporulation, well before SigE (or SigK) is produced or active.

Our results demonstrate that the arrA (S2) transcript is present during exponential growth, when the sporulation-specific sigma factors are not present. Additionally, there is robust transcription from the arrA promoter in vitro with RNA polymerase containing SigA. Further, the sequence of the arrA promoter is close to the consensus sequence of promoters recognized by RNA polymerase containing SigA, and mutations in the conserved −10 region abolished activity of the arrA promoter. Based on these findings, we conclude that arrA is transcribed by RNA polymerase containing SigA and that the phenotypes we observed in the arrA1 mutant are due to loss of arrA transcription that is mediated by RNA polymerase with SigA.

Small RNAs and dnaA in organisms other than B. subtilis

Small RNAs have emerged as a ubiquitous method of gene regulation, especially for the purposes of fine-tuning expression and in stress responses [57–59]. sRNAs have been identified in dnaA and dnaN in a few organisms. For example, there is an antisense RNA with unknown function internal to dnaN in E. coli [22]. An antisense RNA produced from within dnaA in Salmonella enterica appears to increase production of DnaA, perhaps by stabilizing its mRNA [60]. There are two sRNAs in C. crescentus that are predicted to work in trans and target dnaA [61], and a trans-acting sRNA regulates both dnaA and the master cell cycle regulator gcrA in members of the genus Sinorhizobium [62,63]. These examples point to the utility of sRNAs as a method for fine-tuning expression of genes involved in replication and cell cycle control.

DnaA, arrA, sda and sporulation in B. subtilis

Alterations in DnaA activity have profound effects on cellular physiology and development. In addition to acting as the replication initiator, DnaA acts as a transcription factor and in this way, plays an important role in regulating the initiation of sporulation in B. subtilis (and likely other endospore-forming bacteria).

Entry into the sporulation pathway is highly regulated. The key signals to start sporulation include nutrient depletion and high population density [38,64]. Additionally, conditions that alter replication can inhibit the initiation of sporulation [34–36]. These alterations lead to an increase in active DnaA and/or the RecA-dependent host SOS response, both of which cause increased transcription of sda, the product of which is an inhibitor of sporulation initiation. The function of sda is most easily seen following DNA damage or perturbations to replication, both of which cause an increase in the activity of DnaA [9,12,15]. Transcription of sda is also controlled in a cell-cycle-dependent manner and very small changes in the amount of DnaA apparently affect transcription of sda [20].

Our results indicate that modest (~2-fold) changes in the amount of DnaA and DnaN due to arrA affect sporulation initiation. These effects are most likely mediated by DnaA as a direct transcriptional activator of sda. However, we suspect that DnaN might also play a minor role in this. DnaN indirectly affects the activity of DnaA by inhibiting YabA, a protein that normally inhibits cooperative DNA binding by DnaA [29,30]. We suspect that the combined effects of the increase in DnaA and DnaN cause the sda-dependent inhibition of sporulation initiation.

Sda inhibits sporulation primarily by binding to and inhibiting the activity of the kinase KinA [15,17–19]. Based on the effects of: arrA on reducing the amount of DnaA, DnaA on activating transcription of sda, Sda on inhibiting KinA, suppression of the arrA mutant phenotype by loss of sda, and the similarity in phenotypes caused by loss of arrA and a kinA null mutation, the simplest model for our results is that most if not all the effects of arrA on sporulation are mediated by the effects of DnaA activating transcription of Sda (Fig 1B).

Conservation of arrA

arrA appears to be conserved in several Bacillus species as at least 17 have a candidate promoter and associated DnaA binding sites analogous to those in B. subtilis. It is not surprising that arrA is not conserved beyond these species. sRNAs appear to be found in most bacterial species where they have been examined. B. subtilis makes many different sRNAs [21,23,65–67], several of which are antisense to functional genes and known to modulate gene expression [68]. Despite their prevalence, sRNAs are not frequently conserved, even within members of one genus or between strains within the same species. For example, B. licheniformis produces a species-specific sRNA to regulate subtilisin production whereas its close relative B. subtilis does not [69]. Similarly, an RNAseq study of sRNAs in Rhodobacter capsulatus found 422 putative sRNAs, of which 298 appear to be species specific and only 40 have putative inter-taxa homologs [70]. This lack of conservation is thought to reflect the utility of sRNAs as adaptable fine-tuning mechanisms, possibly evolving quickly according to the needs of individual species or strains. Our results highlight the importance of having multiple mechanisms to regulate dnaA, the evolutionary diversity of those mechanisms, and the critical role DnaA plays in the cellular decision to enter into the complex process of sporulation.

Methods

Media and growth conditions

LB medium was used for routine maintenance of both E. coli and B. subtilis. Standard concentrations of antibiotics were used for selection when appropriate [71]. For many experiments (as indicated in the text), B. subtilis cells were grown with shaking at 37°C in S750 defined minimal medium with 50 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer supplemented with 1% glucose, 0.1% glutamate, trace metals, 40 μg/ml phenylalanine, and 40 ug/ml tryptophan [72]. B. subtilis cultures in minimal medium were typically started from cells grown overnight on solid medium to a very light lawn, resuspended and then used to inoculate liquid cultures at OD600 0.05. Cultures grown in LB medium were inoculated with a single colony from a freshly streaked LB agar plate.

Sporulation assays

Cells were grown in Difco Sporulation Medium (DSM) [71]. At the indicated times after entry into stationary phase, samples were diluted and the number of CFUs were determined by plating on LB agar before (total cells) and after (spores) heating to 80°C for 10 min. Percent sporulation was determined by the ratio of spores/ml (CFU/ml after heat treatment) to cells/ml (CFU/ml before heat treatment) x 100%.

Strains and alleles

B. subtilis strains (Table 2) were derived from JH642 (pheA1 trpC2) [73,74] and were constructed by natural transformation.

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Table 2. B. subtilis strains used.

https://doi.org/10.1371/journal.pgen.1011625.t002

arrA1.

arrA1 alters the −10 region of the arrA promoter, changing the 5’-TATAAT to 5’-GGGGGG. It was made by cloning a PCR fragment containing the arrA promoter region into the plasmid pCAL1422 (lacZ, cat) [75], introducing the promoter mutation, and then transforming into B. subtilis selecting for chloramphenicol resistance. Transformants have the plasmid integrated by a single crossover and were lacZ+. A transformant was passaged in LB (without antibiotic) and screened for the loss of lacZ, indicating the loss of the integrated plasmid. The arrA promoter region was PCR amplified from the chromosome of several isolates that had lost lacZ and sequenced to identify a clone that contained the desired mutation in ParrA.

PywlC-lacZ.

A DNA fragment from 240 bp upstream to 73 bp downstream from the start codon of ywlC was PCR-amplified and combined with a chloramphenicol resistance cassette, lacZ, and upstream and downstream homology regions for insertion into amyE using Gibson Isothermal Assembly [76]. These additional components were PCR-amplified using pDG268 as a template [40]. The linear assembly was then integrated into the B. subtilis chromosome at amyE by double crossover, selecting for chloramphenicol resistance.

∆sda::spc.

spc was cloned between sequences flanking sda and then a PCR product containing the allele was introduced into the B. subtilis chromosome by transformation and selection for spectinomycin resistance. The ∆sda::spc allele is missing sda from 40 bp upstream to 142 bp downstream from the start codon of the 138 bp sda open reading frame, essentially as described [15].

SPβ::spoIIA-lacZ.

spoIIA-lacZ was previously described [36,77]. Briefly, it is a transcriptional fusion of the spoIIA promoter region fused to lacZ, integrated into the temperate phage SPβ.

Protein expression and purification

B. subtilis RNA polymerase.

RNA polymerase was purified from the B. subtilis strain MH5636 that expresses rpoC-his10 [78], essentially as described [79], with a few modifications. Briefly, the cell resuspension buffer was supplemented with 5 mM imidazole and β-mercaptoethanol reduced to 2 mM. The extract was clarified using one round of centrifugation followed by filtration through a Whatman FP30/1.2CA filter unit. Talon Superflow resin (Cytiva) was used in place of Ni-NTA, and Heparin and HiQ chromatography was carried out at room temperature, with fractions collected in a 4°C cabinet. The final protein concentration was determined using the Bio-Rad Protein Assay standardized to BSA, and the preparation was stored at −20°C.

Sigma-A.

B. subtilis sigma-A (SigA) protein was isolated from strain JLS116, an E. coli strain previously described [80] that overexpresses a SigA-chitin fusion protein. Purification was carried out essentially as described [80], with minor modifications. Triton X-100 was omitted from the lysis buffer until after lysis by French press was complete. In addition, a protease inhibitor cocktail and 250U of Benzonase nuclease (both from Millipore Sigma) were added immediately after lysis. After centrifugation the lysate was passed through a Whatman FP30/1.2 CA filter unit prior to incubation with the chitin resin. The final preparation was concentrated using a 15-ml Centricon filter (3-kDa cutoff; Amicon), dialyzed against the published storage buffer except that glycerol was increased to 50%, and stored at −20°C. The final protein concentration was determined using the Bio-Rad Protein Assay standardized to BSA.

B. subtilis DnaA.

B. subtilis DnaA was purified as described previously [13].

Templates for in vitro transcription

Templates for in vitro transcription reactions contained DNA from the dnaA/arrA promoter region as indicated in the relevant figures (Figs 2 and 3). Template DNA in each in vitro transcription reaction was a linear PCR product of the indicated region that was made using plasmid DNA as a template for PCR using KAPA HiFi HotStart ReadyMix (Roche Sequencing Solutions). PCR products were purified using a Qiagen MinElute PCR cleanup kit and DNA was eluted in nuclease-free water.

Plasmids pELS33, pELS51, and pELS69 were used to generate the PCR products. pELS33 contains the promoter region from 398 bp upstream to 18 bp downstream and pELS51 and pELS69 contain the promoter region from 547 bp upstream to 18 bp downstream of the dnaA start codon. pELS51 and pELS69 also contain two bidirectional terminators, one at each end of the promoter-containing fragment. Terminators from the gatB/yerO (from 2 bp to 34 bp downstream of the gatB stop codon) and serA/aroC (from 2 bp to 36 bp downstream of the serA stop codon) regions were synthesized as oligonucleotides and cloned into each of the two plasmids via Gibson isothermal assembly [76]. pELS51 and pELS69 are essentially the same, except pELS69 contains the arrA1 mutant promoter (5’-GGGGGG in place of 5’-TATAAT in the −10 region of the promoter).

In vitro transcription

In vitro transcription reactions were done in 20 µl reactions that contained 40 mM HEPES-KOH pH 7.6, 10 mM magnesium acetate, 10 mM sodium chloride, 10 mM potassium chloride, 2% sucrose, 2.5% glycerol, 0.5 mM EDTA, 150 mM potassium glutamate, 1 mM DTT, and 0.2 U/µl RnaseOUT. A mix of nucleotides was prepared than then added to transcription reactions at final concentrations of: 2.5 mM ATP; 0.1 mM each CTP, GTP, and UTP, and 1 µC of alpha-32P-UTP.

The concentrations of DNA template, RNA polymerase, sigma-A, and DnaA-ATP are indicated in figure legends. RNA polymerase, sigma-A, and RNaseOUT were mixed prior to addition to transcription reactions. Where added, DnaA was pre-incubated on ice for 20 minutes with 2.5 mM ATP in DnaA storage buffer (45 mM HEPES-KOH pH 7.6, 10 mM magnesium acetate, 0.5 mM EDTA, 700 mM potassium glutamate, 1 mM DTT). DnaA-ATP (250 nM) was then incubated on ice for 20 minutes with the template DNA and all remaining components listed above. The RNAP mixture was then added to the final concentrations indicated in the figure legends and incubated for another 20 minutes on ice. The reactions were finally started by addition of the nucleotide mix and the reactions were incubated at 37°C for 20 minutes. Reactions were stopped by adding 10 µl of loading buffer (7 M urea, 100 mM EDTA, 4.8% glycerol, 0.25 mg/ml bromophenol blue, 0.25 mg/ml xylene cyanol) and heating to 75°C for 3 minutes. Samples were run on a polyacrylamide gel containing 7 M urea in tris-borate EDTA running buffer. Gels were dried onto Whatman paper and used to expose a Kodak Storage Phosphor Screen SD230 overnight before imaging on a GE Typhoon FLA 9500 scanner.

RNA size standards were transcribed using Century Marker Template Plus fragments (Invitrogen by ThermoFisher Scientific, AM7782) and an Ambion MAXIscript T7 In Vitro Transcription Kit (AM1312) according to the manufacturer’s instructions.

β-galactosidase assays

Cells were grown in defined minimal medium at 37°C to mid-exponential phase, diluted back to OD600 0.05, grown to mid-exponential phase (OD 0.2-0.4) and samples were collected. Growth was stopped and cells permeabilized by addition of toluene (1.5% final concentration). β-galactosidase specific activity ({∆A420 per min per ml of culture per OD600} × 1000) was determined essentially as previously described [81].

RNA isolation and reverse transcription

Cells were grown to mid-exponential phase overnight on solid medium and used to inoculate liquid cultures at OD600 0.05. When cultures reached OD600 0.2-0.5, 1 OD600 of cells was harvested in an equal volume of ice-cold methanol and pelleted. Pellets were resuspended in 200 µl TE pH 8.0 (10 mM Tris-HCl, 1 mM EDTA) with 10 mg/ml lysozyme and incubated for 5 minutes at 37°C. RNA was then isolated using Qiagen RNeasy PLUS kit. Genomic DNA was removed using dsDNase (Thermo Scientific, EN0771). iScript Supermix (Bio-Rad) was used for reverse transcriptase reactions to generate cDNA. RNA was degraded by adding 75% volume of 0.1 M NaOH and incubating at 70°C 10 minutes, then neutralized by adding 75% of the original volume 0.1 M HCl. RNA was normalized on a Thermo Scientific NanoDrop One.

RT-qPCR and qPCR

RT-qPCR and qPCR were done using SsoAdvanced Universal SYBR master mix and CFX96 Touch Real-Time PCR system (Bio-Rad) using indicated primer pairs (S1 Table).

Northern blots

Total RNA for northern blots was purified (as above) from 2-2.5 OD600 units of cells grown in LB medium to mid-exponential phase. Twenty micrograms of RNA in RNA loading dye (New England Biolabs) per lane was run on a 5% acrylamide (19:1 acrylamide:bisacrylamide ratio) gel containing 8M urea and 1xTBE buffer. Size standards (5 µl of Low Range ssRNA Ladder; New England Biolabs) were also run. Gels were stained with SYBR Gold (Invitrogen) and imaged using a UV transilluminator. RNA was transferred to Hybond-N+ membrane (Cytiva) using a BioRad semidry transfer apparatus and then crosslinked using a UV Stratalinker (Stratagene). The membrane was incubated overnight at 42°C in ULTRAhyb Ultrasensitive Hybridization Buffer (Invitrogen) with an oligonucleotide (oJLS184; 5’-cacagcttgtgtagaaggttgtccacaag) that was end-labeled (T4 polynucleotide kinase, New England Biolabs) with ATP [γ−32P] (PerkinElmer). This oligonucleotide is complementary to the 5’ portion of the arrA transcript (Fig 4A). The membrane was washed at 42°C in 2X SSC, 0.5% SDS, exposed to a phosphorstorage screen (GE Life Science) and imaged with a laser scanner (Typhoon FLA9500, GE Life Sciences).

DNA isolation

Cultures were grown to mid-exponential phase (OD600 0.2-0.4) in defined minimal medium at 37°C. Cells were harvested in one volume of ice-cold methanol, pelleted, and stored at −80°C. Genomic DNA was isolated using Qiagen DNeasy kit with 40 μg/ml lysozyme.

Western blots

Cultures were grown to mid-exponential phase overnight on solid medium and used to inoculate liquid cultures at OD600 0.05. When cultures reached OD600 0.2-0.5, 1 OD600 of cells was harvested and lysed with 1µg lysozyme and P8849 protease inhibitor cocktail at 37°C. Due to the difficulty of accurately measuring protein concentration following addition of lysozyme, lysates were normalized to culture OD and mixed with ⅓ volume of 4X protein loading buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol, 20% β-mercaptoethanol, 0.002% bromophenol blue). Equal volumes of each normalized lysate from four biological replicates were separated on a single 10% polyacrylamide gel and transferred to a nitrocellulose membrane using the Trans-blot SD semi-dry transfer cell (Biorad). The membrane was washed in water and then incubated in Revert 700 Total Protein Stain. After washing with Revert 700 Wash Solution, the stained blot was imaged on a LiCor scanner. After imaging, the blot was washed in water before proceeding with western blotting. According to the manufacturer’s instructions, blots were blocked with ½ X Odyssey Blocking Buffer (diluted 1:1 in phosphate-buffered saline) for 1 hr at room temperature. Blots were then incubated with primary antibody (rabbit 1:10,000 in ½ X Odyssey Blocking Buffer with 0.2% Tween) overnight at 4°C. The primary antibodies were rabbit polyclonal antibodies made against purified DnaA and DnaN (Covance). Blots were washed 5 times with phosphate-buffered saline + 0.2% Tween for at least 5 min each and then incubated for 30 min at room temperature with secondary antibody (LiCor dye 800 goat anti-rabbit 1:10,000 in ½ X Odyssey Blocking Buffer + 0.2% Tween + 0.01% SDS). Blots (S1 Image in S1 File) were imaged and quantitated on a LiCor scanner.

Quantitation of total protein, DnaA, and DnaN was performed in ImageJ using band densitometry. Each DnaA and DnaN band was normalized to the total protein signal between 250 kD and 20 kD in their respective lanes. DnaA bands were normalized to the average of all wildtype DnaA bands in order to display the fold change between wild type and the arrA1 mutant. DnaN was normalized similarly.

Search for a possible antisense promoter and DnaA binding sites between dnaA and its promoter

Reference genomes were accessed through the NCBI National Library of Medicine Genome Browser. The search term “Bacillus (Bacillus rRNA group 1)” was entered into the taxonomic search bar and filtered by Status and Assembly Level to show only reference genomes assembled to the chromosome level or beyond. Metadata for 37 “Bacillus” results were downloaded along with RefSeq files (Sequences and Annotations in GBFF format). Reference genomes B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii were omitted as they are identical to our wildtype strain, AG174, in the regions compared. We extracted the antisense sequence of the intergenic region between dnaA and rpmH from each file, aligned them using the ClustalO algorithm in JalView. We then looked for conservation of the DnaA binding sites and scanned the alignment for possible promoters, analogous to those present in B. subtilis and B. licheniformis.

Supporting information

S1 Table. Primers for RT-qPCR and qPCR1.

List of primers used for RT-qPCR and pPCR, including primers for measuring mRNA from dnaA, dnaN, and gyrA (for normalization) and for determining the ori to ter ratio.

https://doi.org/10.1371/journal.pgen.1011625.s001

(PDF)

S2 Table. Reference genomes.

Table includes accession numbers, species, and strain information for genomes that were analyzed for possible promoters and DnaA binding sites between the dnaA promoter and open reading frame, similar to those found in B. subtilis and B. licheniformis.

https://doi.org/10.1371/journal.pgen.1011625.s002

(PDF)

S1 Fig. The amount of DnaA and DnaN is increased in the arrA1 mutant early in sporulation.

Wild type (AG174) and arrA1 mutant (ELS393) cells were grown in sporulation medium (DSM) and the amount of (A) DnaA and (B) DnaN was measured by western blotting at the times indicated. Time 0 (T0) represents entry into stationary phase. The OD600 of the culture at the time of sampling was used to load equivalent amounts of lysate in each lane. Three independent experiments were performed for both strains and the amount of each protein was normalized to that in wild type cells at T0 for each experiment. The average for each time point ± the standard error of the mean is presented.

https://doi.org/10.1371/journal.pgen.1011625.s003

(TIF)

S1 Data. Underlying raw data.

The excel spreadsheet contains the underlying data for experiments presented in the indicated figures and Table 1.

https://doi.org/10.1371/journal.pgen.1011625.s004

(XLSX)

S1 File.

Images. Images of gels and blots used in Figs 4 and 5. S1A. Images of the northern blot (left) and stained gel (right) that were used for Fig 4A and 4B, respectively. S1B. Images of the western blot (top) and stained gel (bottom) that were used for Fig 5B.

https://doi.org/10.1371/journal.pgen.1011625.s005

(PDF)

Acknowledgments

We thank Mary Anderson for helpful discussions and comments, and Peter Zuber and Cierra Birch for strains and advice on purifying RNA polymerase and Sigma-A.

References

  1. 1. Kaguni JM. DnaA: controlling the initiation of bacterial DNA replication and more. Annu Rev Microbiol. 2006;60:351–75. pmid:16753031
  2. 2. Menikpurage IP, Woo K, Mera PE. Transcriptional Activity of the Bacterial Replication Initiator DnaA. Front Microbiol. 2021;12:662317. pmid:34140937
  3. 3. Mott ML, Berger JM. DNA replication initiation: mechanisms and regulation in bacteria. Nat Rev Microbiol. 2007;5(5):343–54. pmid:17435790
  4. 4. Chodavarapu S, Kaguni JM. Replication Initiation in Bacteria. Enzymes. 2016;39:1–30. pmid:27241926
  5. 5. Rashid F, Berger JM. How bacteria initiate DNA replication comes into focus. Bioessays. 2025;47(1):e2400151. pmid:39390825
  6. 6. Jameson KH, Wilkinson AJ. Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli. Genes (Basel). 2017;8(1):22. pmid:28075389
  7. 7. Felletti M, Omnus DJ, Jonas K. Regulation of the replication initiator DnaA in Caulobacter crescentus. Biochim Biophys Acta Gene Regul Mech. 2019;1862(7):697–705. pmid:29382570
  8. 8. Goranov AI, Katz L, Breier AM, Burge CB, Grossman AD. A transcriptional response to replication status mediated by the conserved bacterial replication protein DnaA. Proc Natl Acad Sci U S A. 2005;102(36):12932–7. pmid:16120674
  9. 9. Ishikawa S, Ogura Y, Yoshimura M, Okumura H, Cho E, Kawai Y, et al. Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method. DNA Res. 2007;14(4):155–68. pmid:17932079
  10. 10. Cho E, Ogasawara N, Ishikawa S. The functional analysis of YabA, which interacts with DnaA and regulates initiation of chromosome replication in Bacillus subtils. Genes Genet Syst. 2008;83(2):111–25. pmid:18506095
  11. 11. Breier AM, Grossman AD. Dynamic association of the replication initiator and transcription factor DnaA with the Bacillus subtilis chromosome during replication stress. J Bacteriol. 2009;191(2):486–93. pmid:19011033
  12. 12. Hoover SE, Xu W, Xiao W, Burkholder WF. Changes in DnaA-dependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium. J Bacteriol. 2010;192(15):3915–24. pmid:20511500
  13. 13. Smith JL, Grossman AD. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA. PLoS Genet. 2015;11(5):e1005258. pmid:26020636
  14. 14. Seid CA, Smith JL, Grossman AD. Genetic and biochemical interactions between the bacterial replication initiator DnaA and the nucleoid-associated protein Rok in Bacillus subtilis. Mol Microbiol. 2016;103(5):798–817. pmid:27902860
  15. 15. Burkholder WF, Kurtser I, Grossman AD. Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Cell. 2001;104(2):269–79. pmid:11207367
  16. 16. Washington TA, Smith JL, Grossman AD. Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis. Mol Microbiol. 2017;106(1):109–28. pmid:28752667
  17. 17. Rowland SL, Burkholder WF, Cunningham KA, Maciejewski MW, Grossman AD, King GF. Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis. Mol Cell. 2004;13(5):689–701. pmid:15023339
  18. 18. Whitten AE, Jacques DA, Hammouda B, Hanley T, King GF, Guss JM, et al. The structure of the KinA-Sda complex suggests an allosteric mechanism of histidine kinase inhibition. J Mol Biol. 2007;368(2):407–20. pmid:17350039
  19. 19. Cunningham KA, Burkholder WF. The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F. Mol Microbiol. 2009;71(3):659–77. pmid:19040634
  20. 20. Veening J-W, Murray H, Errington J. A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. Genes Dev. 2009;23(16):1959–70. pmid:19684115
  21. 21. Nicolas P, Mäder U, Dervyn E, Rochat T, Leduc A, Pigeonneau N, et al. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science. 2012;335(6072):1103–6. pmid:22383849
  22. 22. Lalanne J-B, Taggart JC, Guo MS, Herzel L, Schieler A, Li G-W. Evolutionary Convergence of Pathway-Specific Enzyme Expression Stoichiometry. Cell. 2018;173(3):749-761.e38. pmid:29606352
  23. 23. Lawaetz AC, Cowley LA, Denham EL. Genome-wide annotation of transcript boundaries using bacterial Rend-seq datasets. Microb Genom. 2024;10(4):001239. pmid:38668652
  24. 24. Ogura Y, Imai Y, Ogasawara N, Moriya S. Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis. J Bacteriol. 2001;183(13):3833–41. pmid:11395445
  25. 25. Goranov AI, Breier AM, Merrikh H, Grossman AD. YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress. Mol Microbiol. 2009;74(2):454–66. pmid:19737352
  26. 26. Noirot-Gros M-F, Dervyn E, Wu LJ, Mervelet P, Errington J, Ehrlich SD, et al. An expanded view of bacterial DNA replication. Proc Natl Acad Sci U S A. 2002;99(12):8342–7. pmid:12060778
  27. 27. Noirot-Gros M-F, Velten M, Yoshimura M, McGovern S, Morimoto T, Ehrlich SD, et al. Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis. Proc Natl Acad Sci U S A. 2006;103(7):2368–73. pmid:16461910
  28. 28. Hayashi M, Ogura Y, Harry EJ, Ogasawara N, Moriya S. Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation. FEMS Microbiol Lett. 2005;247(1):73–9. pmid:15927750
  29. 29. Merrikh H, Grossman AD. Control of the replication initiator DnaA by an anti-cooperativity factor. Mol Microbiol. 2011;82(2):434–46. pmid:21895792
  30. 30. Scholefield G, Murray H. YabA and DnaD inhibit helix assembly of the DNA replication initiation protein DnaA. Mol Microbiol. 2013;90(1):147–59. pmid:23909787
  31. 31. Anderson ME, Smith JL, Grossman AD. Multiple mechanisms for overcoming lethal over-initiation of DNA replication. Mol Microbiol. 2022;118(4):426–42. pmid:36053906
  32. 32. Reed TT, Kendal AH, Wozniak KJ, Simmons LA. DNA replication initiation timing is important for maintaining genome integrity. bioRxiv. 2024;2024.06.18.599555. pmid:38948856
  33. 33. Moriya S, Kato K, Yoshikawa H, Ogasawara N. Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells’ initiation potential. EMBO J. 1990;9(9):2905–10. pmid:2167836
  34. 34. Lemon KP, Kurtser I, Wu J, Grossman AD. Control of initiation of sporulation by replication initiation genes in Bacillus subtilis. J Bacteriol. 2000;182(10):2989–91. pmid:10781575
  35. 35. Ireton K, Grossman AD. A developmental checkpoint couples the initiation of sporulation to DNA replication in Bacillus subtilis. EMBO J. 1994;13(7):1566–73. pmid:8156995
  36. 36. Ireton K, Grossman AD. Coupling between gene expression and DNA synthesis early during development in Bacillus subtilis. Proc Natl Acad Sci U S A. 1992;89(18):8808–12. pmid:1528896
  37. 37. Phillips ZEV, Strauch MA. Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci. 2002;59(3):392–402. pmid:11964117
  38. 38. Piggot PJ, Hilbert DW. Sporulation of Bacillus subtilis. Curr Opin Microbiol. 2004;7(6):579–86. pmid:15556029
  39. 39. Perego M, Cole SP, Burbulys D, Trach K, Hoch JA. Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis. J Bacteriol. 1989;171(11):6187–96. pmid:2509430
  40. 40. Antoniewski C, Savelli B, Stragier P. The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. J Bacteriol. 1990;172(1):86–93. pmid:2104615
  41. 41. Pichon C, Felden B. Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains. Proc Natl Acad Sci U S A. 2005;102(40):14249–54. pmid:16183745
  42. 42. Mraheil MA, Billion A, Kuenne C, Pischimarov J, Kreikemeyer B, Engelmann S, et al. Comparative genome-wide analysis of small RNAs of major Gram-positive pathogens: from identification to application. Microb Biotechnol. 2010;3(6):658–76. pmid:21255362
  43. 43. Tsui H-CT, Mukherjee D, Ray VA, Sham L-T, Feig AL, Winkler ME. Identification and characterization of noncoding small RNAs in Streptococcus pneumoniae serotype 2 strain D39. J Bacteriol. 2010;192(1):264–79. pmid:19854910
  44. 44. Shioya K, Michaux C, Kuenne C, Hain T, Verneuil N, Budin-Verneuil A, et al. Genome-wide identification of small RNAs in the opportunistic pathogen Enterococcus faecalis V583. PLoS One. 2011;6(9):e23948. pmid:21912655
  45. 45. Acebo P, Martin-Galiano AJ, Navarro S, Zaballos A, Amblar M. Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae. RNA. 2012;18(3):530–46. pmid:22274957
  46. 46. Mann B, van Opijnen T, Wang J, Obert C, Wang Y-D, Carter R, et al. Control of virulence by small RNAs in Streptococcus pneumoniae. PLoS Pathog. 2012;8(7):e1002788. pmid:22807675
  47. 47. Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, et al. Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS Genet. 2013;9(5):e1003493. pmid:23675309
  48. 48. Bronsard J, Pascreau G, Sassi M, Mauro T, Augagneur Y, Felden B. sRNA and cis-antisense sRNA identification in Staphylococcus aureus highlights an unusual sRNA gene cluster with one encoding a secreted peptide. Sci Rep. 2017;7(1):4565. pmid:28676719
  49. 49. Sinel C, Augagneur Y, Sassi M, Bronsard J, Cacaci M, Guérin F, et al. Small RNAs in vancomycin-resistant Enterococcus faecium involved in daptomycin response and resistance. Sci Rep. 2017;7(1):11067. pmid:28894187
  50. 50. Sinha D, Zimmer K, Cameron TA, Rusch DB, Winkler ME, De Lay NR. Redefining the Small Regulatory RNA Transcriptome in Streptococcus pneumoniae Serotype 2 Strain D39. J Bacteriol. 2019;201(14):e00764-18. pmid:30833353
  51. 51. Subramanian D, Bhasuran B, Natarajan J. Genomic analysis of RNA-Seq and sRNA-Seq data identifies potential regulatory sRNAs and their functional roles in Staphylococcus aureus. Genomics. 2019;111(6):1431–46. pmid:30304708
  52. 52. Wiegand S, Dietrich S, Hertel R, Bongaerts J, Evers S, Volland S, et al. RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation. BMC Genomics. 2013;14:667. pmid:24079885
  53. 53. Guo J, Cheng G, Gou X-Y, Xing F, Li S, Han Y-C, et al. Comprehensive transcriptome and improved genome annotation of Bacillus licheniformis WX-02. FEBS Lett. 2015;589(18):2372–81. pmid:26226425
  54. 54. Gorbatyuk B, Marczynski GT. Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus. Mol Microbiol. 2005;55(4):1233–45. pmid:15686567
  55. 55. Cheng L, Keiler KC. Correct timing of dnaA transcription and initiation of DNA replication requires trans translation. J Bacteriol. 2009;191(13):4268–75. pmid:19429626
  56. 56. Mars RAT, Nicolas P, Denham EL, van Dijl JM. Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression. Microbiol Mol Biol Rev. 2016;80(4):1029–57. pmid:27784798
  57. 57. Jørgensen MG, Pettersen JS, Kallipolitis BH. sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms. Biochim Biophys Acta Gene Regul Mech. 2020;1863(5):194504. pmid:32061884
  58. 58. Adams PP, Storz G. Prevalence of small base-pairing RNAs derived from diverse genomic loci. Biochim Biophys Acta Gene Regul Mech. 2020;1863(7):194524. pmid:32147527
  59. 59. Papenfort K, Melamed S. Small RNAs, Large Networks: Posttranscriptional Regulons in Gram-Negative Bacteria. Annu Rev Microbiol. 2023;77:23–43. pmid:36944261
  60. 60. Dadzie I, Xu S, Ni B, Zhang X, Zhang H, Sheng X, et al. Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi. PLoS One. 2013;8(4):e61308. pmid:23637809
  61. 61. Beroual W, Brilli M, Biondi EG. Non-coding RNAs Potentially Controlling Cell Cycle in the Model Caulobacter crescentus: A Bioinformatic Approach. Front Genet. 2018;9:164. pmid:29899753
  62. 62. Jiménez-Zurdo JI, Robledo M. Unraveling the universe of small RNA regulators in the legume symbiont Sinorhizobium meliloti. Symbiosis. 2015;67(1–3):43–54.
  63. 63. Robledo M, Jiménez-Zurdo JI, Becker A. Antisense transcription of symbiotic genes in Sinorhizobium meliloti. Symbiosis. 2015;67(1–3):55–67.
  64. 64. Grossman AD. Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu Rev Genet. 1995;29:477–508. pmid:8825484
  65. 65. Irnov I, Sharma CM, Vogel J, Winkler WC. Identification of regulatory RNAs in Bacillus subtilis. Nucleic Acids Res. 2010;38(19):6637–51. pmid:20525796
  66. 66. Dambach M, Irnov I, Winkler WC. Association of RNAs with Bacillus subtilis Hfq. PLoS One. 2013;8(2):e55156. pmid:23457461
  67. 67. Brantl S, Brückner R. Small regulatory RNAs from low-GC Gram-positive bacteria. RNA Biol. 2014;11(5):443–56. pmid:24576839
  68. 68. Brantl S, Müller P. Cis- and Trans-Encoded Small Regulatory RNAs in Bacillus subtilis. Microorganisms. 2021;9(9):1865. pmid:34576762
  69. 69. Hertel R, Meyerjürgens S, Voigt B, Liesegang H, Volland S. Small RNA mediated repression of subtilisin production in Bacillus licheniformis. Sci Rep. 2017;7(1):5699. pmid:28720814
  70. 70. Grüll MP, Peña-Castillo L, Mulligan ME, Lang AS. Genome-wide identification and characterization of small RNAs in Rhodobacter capsulatus and identification of small RNAs affected by loss of the response regulator CtrA. RNA Biol. 2017;14(7):914–25. pmid:28296577
  71. 71. Harwood CR, Cutting SM, editors. Molecular biological methods for Bacillus. Chichester. New York: Wiley; 1990.
    • 72. Jaacks KJ, Healy J, Losick R, Grossman AD. Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis. J Bacteriol. 1989;171(8):4121–9. pmid:2502532
    • 73. Perego M, Spiegelman GB, Hoch JA. Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis. Mol Microbiol. 1988;2(6):689–99. pmid:3145384
    • 74. Smith JL, Goldberg JM, Grossman AD. Complete Genome Sequences of Bacillus subtilis subsp. subtilis Laboratory Strains JH642 (AG174) and AG1839. Genome Announc. 2014;2(4):e00663-14. pmid:24994804
    • 75. Thomas J, Lee CA, Grossman AD. A conserved helicase processivity factor is needed for conjugation and replication of an integrative and conjugative element. PLoS Genet. 2013;9(1):e1003198. pmid:23326247
    • 76. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343–5. pmid:19363495
    • 77. Wu JJ, Piggot PJ, Tatti KM, Moran CP Jr. Transcription of the Bacillus subtilis spoIIA locus. Gene. 1991;101(1):113–6. pmid:1905664
    • 78. Qi Y, Hulett FM. PhoP-P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP-P activator sites within the coding region stimulate transcription in vitro. Mol Microbiol. 1998;28(6):1187–97. pmid:9680208
    • 79. Birch CA, Davis MJ, Mbengi L, Zuber P. Exploring the Amino Acid Residue Requirements of the RNA Polymerase (RNAP) α Subunit C-Terminal Domain for Productive Interaction between Spx and RNAP of Bacillus subtilis. J Bacteriol. 2017;199(14):e00124-17. pmid:28484046
    • 80. Nakano MM, Geng H, Nakano S, Kobayashi K. The nitric oxide-responsive regulator NsrR controls ResDE-dependent gene expression. J Bacteriol. 2006;188(16):5878–87. pmid:16885456
    • 81. Miller J. Experiments in molecular genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1972.