The Bacillus subtilis Late Competence Operon comE Is Transcriptionally Regulated by yutB and under Post-Transcription Initiation Control by comN (yrzD)

Mitsuo Ogura; Teruo Tanaka

doi:10.1128/JB.01429-08

. 2008 Nov 21;191(3):949–958. doi: 10.1128/JB.01429-08

The Bacillus subtilis Late Competence Operon comE Is Transcriptionally Regulated by yutB and under Post-Transcription Initiation Control by comN (yrzD) ^▿^†

Mitsuo Ogura ^1,^*, Teruo Tanaka ¹

PMCID: PMC2632086 PMID: 19028902

Abstract

The Bacillus subtilis genome has been sequenced, and disruptants with disruptions in genes that were not characterized previously were systematically generated. We screened these gene disruptants for decreased transformation frequency and identified two genes, yrzD and yutB, whose disruption resulted in severely reduced transformation frequency and modestly reduced transformation frequency, respectively. In the regulation of competence development, various signals affect the expression of comK, which encodes a master regulator of genetic competence that drives late competence gene transcription. Epistatic analyses of both the yrzD and yutB genes revealed no significant differences in the expression of comK. Further analysis of the expression of late competence genes in the yrzD disruptant revealed that yrzD is specifically required for regulation of the comE operon, which is one of the late competence operons, and thus was renamed comN. An analysis of various comE-lacZ fusions revealed that the target cis element for comN action is in the large (approximately 1-kb) 5′ untranslated region of comE, while the activity of the comE promoter was not affected by disruption of comN. These results suggested that there is post-transcription initiation control of comE by comN. A sequential deletion analysis of this region revealed the 35-bp region required for comN action. The yutB gene encodes a putative lipoic acid synthetase and yet is specifically required for transcription of comE, based on the results of lacZ fusion analyses. Therefore, yutB and comN regulate comE at the transcription and post-transcription initiation levels, respectively. These results demonstrate that a comE-specific regulatory mechanism is involved in development of genetic competence.

Lateral gene transfer has played an important role in the evolution of bacteria, accounting for such developments as gain of pathogenic islands and antibiotic resistance (7, 33). One of the modes of lateral gene transfer is natural genetic competence (7). Genetic competence in the gram-positive bacterium Bacillus subtilis develops when cells are grown in glucose-based minimal medium during the early stationary phase and a subpopulation of the cells differentiates into competent cells (12).

The main components of the regulatory cascade leading to competence development in B. subtilis have been elucidated (13). The pheromone ComX is a cell density signal and triggers autophosphorylation of ComP, the sensor kinase in the ComP-ComA two-component system. Subsequent transfer of a phosphoryl group from ComP to ComA, the response regulator, results in activation of ComA and thus expression of the srfA operon. The srfA operon contains the biosynthetic genes for the biosurfactant surfactin (11). RapC, RapD, RapF, RapG, and RapH inhibit expression of the ComA regulon (3, 10, 20, 34, 42), and their cognate extracellular Phr pentapeptide ligands are internalized and inhibit Rap activity. Thus, the Rap-Phr system is a regulatory system for the expression of srfA. Activation of ComK requires ComS, which is encoded by another open reading frame (ORF) in the srfA transcript. ComS mediates the release of ComK from the MecA-ClpCP-ComK complex (by proteolysis with ClpCP), leading to autoactivation of ComK (13).

ComK autoactivation is important for bistability in competent and noncompetent cells (30, 41). Additional activators, such as DegU, and repressors, including CodY, AbrB, and Rok, are also involved in ComK activation. Phosphorylated Spo0A, which is a master regulator of the initiation of sporulation, inhibits abrB transcription, leading to the release of comK from AbrB-mediated repression (13). ComK is a transcriptional regulatory protein that activates the expression of many genes, including late competence operons encoding the protein components needed for uptake and processing of foreign DNA (2, 18, 37). For example, ComEA, ComEC, and ComFA are a DNA-binding receptor, a membrane channel, and a DNA translocase, respectively (7, 16, 22, 29, 39). The comE operon has an unusual operon structure that notably includes an approximately 1-kb 5′ untranslated region (5′-UTR) (16). Another ComK-regulated operon, the comG operon, contains seven ORFs, and ComGA is a putative ATPase (1, 7). The pilin-like protein ComGC, which is processed by the peptidase ComC, assembles to form a multimeric structure in the cell surface (7, 8). In addition to activation of the late competence genes, ComK is required for the activation of unknown genes responsible for the localization of ComGA, ComFA, and the single-stranded DNA-binding protein YwpH to the poles of the bacterium (17). Localization studies using green fluorescent protein have revealed that there is a close relationship between the protein components of the competence machinery and the recombination system. For example, it has been shown that ComGA and extended RecA filaments are closely associated (23). At the cell poles, these proteins form a multiprotein complex for transformation (8, 25).

The B. subtilis genome has been sequenced (27), and disruptants with disruptions in the genes that had not been characterized yet were systematically generated (24). In the present study, we screened these gene disruptants for decreased transformation frequency and identified two genes, yrzD and yutB, whose disruption resulted in reduced competence without a significant effect on comK. The yrzD gene is required for post-transcription initiation control of the comE operon and thus was renamed comN. The target cis element for ComN activity is in the 5′-UTR of the comE operon. The yutB gene encodes a putative lipoic acid synthetase and is required for transcription of the comE operon.

MATERIALS AND METHODS

Bacterial strains and culture media.

All the B. subtilis strains used in this study are listed in Table 1. One-step competence medium (MC) (26), Antibiotic III medium, and LB medium (Difco, Lawrence, KS) were used. The concentrations of antibiotics used have been described previously (36).

TABLE 1.

B. subtilis strains and plasmids used in this study

Strain or plasmid	Genotype or description	Source and/or reference
Strains
168	trpC2	Laboratory stock
YUTBd	trpC2 yutB::Em^r (yutB-lacZ)	BSORF^a
YRZDc	trpC2 comN::Cm^r	BSORF^a
OAM304	trpC2 yutB::Pm^r	This study
OAM434	trpC2 comN::Cm^ramyE::comN (Sp^r)	This study
OAM435	trpC2 yutB::Pm^ramyE::yutB (Sp^r)	This study
8G32	trpC2 comK (Km^r)	46
8G33	trpC2 comK-lacZ (Km^r)	46
OAM331	trpC2 comK-lacZ (Km^r) comN::Cm^r	This study
OAM332	trpC2 comK-lacZ (Km^r) yutB::Pm^r	This study
OAM236	trpC2 comG-lacZ (Cm^r)	Original background, BD630 (1)
OAM335	trpC2 comG-lacZ (Cm^r::Tc^r)	This study
OAM337	trpC2 comG-lacZ (Cm^r::Tc^r) comN::Cm^r	This study
OAM338	trpC2 comG-lacZ (Cm^r::Tc^r) yutB::Pm^r	This study
OAM339	trpC2 comF-lacZ (Cm^r)	Original background, BD630 (29)
OAM340	trpC2 comF-lacZ (Cm^r::Tc^r)	This study
OAM342	trpC2 comF-lacZ (Cm^r::Tc^r) comN::Cm^r	This study
OAM343	trpC2 comF-lacZ (Cm^r::Tc^r) yutB::Pm^r	This study
OAM344	trpC2 comEA-lacZ (Em^r)	Original background, CU741 (35)^b
OAM346	trpC2 comEA-lacZ (Em^r) comN::Cm^r	This study
OAM347	trpC2 comEA-lacZ (Em^r) yutB::Pm^r	This study
OAM348	trpC2 comC-lacZ (Em^r)	Original background, CU741 (35)
OAM350	trpC2 comC-lacZ (Em^r) comN::Cm^r	This study
OAM351	trpC2 comC-lacZ (Em^r) yutB::Pm^r	This study
OAM436	trpC2 amyE::comEA5-lacZ (Cm^r::Tc^r)	This study
OAM437	trpC2 amyE::comEA5-lacZ (Cm^r::Tc^r) comN::Cm^r	This study
OAM445	trpC2 amyE::comEA5-lacZ (Cm^r::Tc^r) yutB::Pm^r	This study
OAM438	trpC2 comEA2-lacZ (Em^r)	This study
OAM439	trpC2 comEA2-lacZ (Em^r) comN::Cm^r	This study
OAM457	trpC2 comEA21-lacZ (Em^r)	This study
OAM458	trpC2 comEA21-lacZ (Em^r) comN::Cm^r	This study
OAM459	trpC2 comEA2a-lacZ (Em^r)	This study
OAM460	trpC2 comEA2a-lacZ (Em^r) comN::Cm^r	This study
OAM461	trpC2 comEA2b-lacZ (Em^r)	This study
OAM462	trpC2 comEA2b-lacZ (Em^r) comN::Cm^r	This study
OAM440	trpC2 comEA3-lacZ (Em^r)	This study
OAM441	trpC2 comEA3-lacZ (Em^r) comN::Cm^r	This study
OAM442	trpC2 comEA4-lacZ (Em^r)	This study
OAM443	trpC2 comEA4-lacZ (Em^r) comN::Cm^r	This study
OAM444	trpC2 comN-lacZ (Em^r)	This study
OAM549	trpC2 rapC (Em^r-lacZ::Tc^rlacI )	19
OAM550	trpC2 amyE::P_T5-lacZ (Cm^r::Tc^r) rapC (Em^r-lacZ::Tc^rlacI)	This study
OAM546	trpC2 amyE::P_T5-lacZ (Cm^r::Tc^r) rapC (Em^r-lacZ::Tc^rlacI) comN::Cm^r	This study
OAM547	trpC2 amyE::P_T5-UTR-lacZ (Cm^r::Tc^r) rapC (Em^r-lacZ::Tc^rlacI)	This study
OAM548	trpC2 amyE::P_T5-UTR-lacZ (Cm^r Tc^r) rapC (Em^r-lacZ::Tc^rlacI) comN::Cm^r	This study
Plasmids
pDG1730	Insertion vector for amyE, spectinomycin and erythromycin resistance	15
pDG1730-comN	pDG1730 carrying comN (position −181 relative to the first codon to the stop codon of comN)	This study
pDG1730-yutB	pDG1730 carrying yutB (position −180 relative to the first codon to the stop codon of yutB)	This study
pPhl2	Insertion vector, pheomycin resistance	36
pPhl-yutB	pPhl2 carrying part of yutB	This study
pMutinIII	Insertion vecter, ampicillin and erythromycin resistance, lacZI	45
pMut-comEA	pMutinIII carrying part of 5′-UTR of comEA (positions +879 to +1177 relative to the transcription start site of comE)	35
pMut-comEA2	pMutinIII carrying part of 5′-UTR of comEA (positions +504 to +858 relative to the transcription start site of comE)	This study
pMut-comEA21	pMutinIII carrying part of 5′-UTR of comEA (positions +504 to +823 relative to the transcription start site of comE)	This study
pMut-comEA2a	pMutinIII carrying part of 5′-UTR of comEA (positions +259 to +758 relative to the transcription start site of comE)	This study
pMut-comEA2b	pMutinIII carrying part of 5′-UTR of comEA (positions +259 to +658 relative to the transcription start site of comE)	This study
pMut-comEA4	pMutinIII carrying part of 5′-UTR of comEA (positions +59 to +358 relative to the transcription start site of comE)	This study
pMut-comN	pMutinIII carrying part of comN	This study
pIS284	Insertion vector for amyE, chloramphenicol resistance, lacZ	44
pIS-comE	pIS284 carrying an upstream region of come (positions −242 to 3 relative to the transcription start site of comE)	This study
pQE8	Ampicillin resistance, P_T5 promoter	Qiagen
pIS-PT5	pIS284 carrying a P_T5 promoter	This study
pIS-PT5-UTR	pIS284 carrying a P_T5 promoter and part of comE UTR	This study
pRB373	Kanamycin and ampicillin resistance	5
pRB-comN	pRB373 carrying comN	This study
ECE75	Ampicillin resistance, Cm^r::Tc^r	43

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http://bacillus.genome.jp/.

We previously stated that this fusion was constructed by cloning a PCR product generated by using oligonucleotides comE-U and comE-D into pMutinIII (35); however, the correct oligonucleotides are comE-H and comE-Bg (Table 2).

Screening of mutants with decreased transformation frequency.

The first round of screening was performed as described previously (38). The donor DNA used was total DNA or plasmid DNA.

Transformation assay.

The transformation assay was carried out as follows. Total DNA or plasmid DNA containing an appropriate antibiotic resistance gene was added to cell cultures 2 h after entry into the stationary phase. After transformation, the cultures were subjected to serial 10-fold dilution. Each diluted fraction was then plated onto three LB agar plates containing appropriate antibiotics, and colonies were counted. The numbers of viable cells were determined by plating a culture onto three LB agar plates containing appropriate antibiotics after 10⁻⁵ dilution.

Strain construction.

YUTBd was constructed by the plasmid insertion method described previously (24, 45). YRZDc was constructed using the PCR-based method described previously, and the oligonucleotides used are shown in Table 2 (20, 24).

TABLE 2.

Oligonucleotides used in this study

Oligonucleotide	Sequence	Product
yutB-Sa	5′-ATGGTCGACCGCGTGTTTCAAATTC-3′	pPhl-yutB
yutB-E	5′-ATGGAATTCAGAAAGCCCGAATGGCT-3′	pPhl-yutB
comE-Bg	5′-ATCAGATCTCCCGCTTTCTCAATTGCCTG-3′	pMut-comEA
comE-H	5′-CTTAAGCTTATGACGAAAGCGGGGAACAT-3′	pMut-comEA
yrzD-F1	5′-CCTGTTCAAGGACTTGTGAC-3′	YRZDc
yrzD-R1	5′-GTTATCCGCTCACAATTCCTCCACGAATTCCACGCTCC-3′	YRZDc
yrzD-F2	5′-CGTCGTGACTGGGAAAACAGCAGGTTGATTCGCACAGG-3′	YRZDc
yrzD-R2	5′-ACCAAATGACAAGCTGGTTG-3′	YRZDc
comE-H2	5′-GATAAGCTTGTTCGAGCTGTTCGGTTT-3′	pMut-comEA2
comE-B2	5′-ATCGGATCCATGGTTTAGAGGATAATAGC-3′	pMut-comEA2
comE-B2b	5′-ATCGGATCCAGTCTCGAAAAGCTCGTTTC-3′	pMut-comEA2b
comE-B2a	5′-ATCGGATCCATCAAAAGCGGCCGATCCC-3′	pMut-comEA2a
comE-B21	5′-ATCGGATCCATTGAAGATAGGCTTTAT-3′	pMut-comEA21
comE-H3	5′-GATAAGCTTCAGCTGCATCTATAAACCG-3′	pMut-comEA3
comE-B3	5′-ATCGGATCCAAGATCATATTCTCATTTC-3′	pMut-comEA3
comE-H4	5′-GATAAGCTTGAAATACCCGGTGAAACA-3′	pMut-comEA4
comE-B4	5′-ATCGGATCCTGCAAATTGAAAGTGACAT-3′	pMut-comEA4
yrzD-Mut-H	5′-CGCAAGCTTAGCCCGACTCCTTCTTCACC-3′	pMut-comN
yrzD-Mut-B	5′-AGCGGATCCGAATTCCACGCTCCTTTTTTA-3′	pMut-comN
yrzD-H	5′-ATCAAGCTTCTATCTCAGCAGTTCTTCCA-3′	pDG1730-comN
yrzD-op-Ba	5′-ATCGGATCCATTCTTGTAACCATTCCTGC-3′	pDG1730-comN
yutB-op-Ba	5′-ATCGGATCCAATGTCAGTAATCATCTTCC-3′	pDG1730-yutB
yutB-op-Hi	5′-ATCAAGCTTATGCTTGTGCTTGACGCT-3′	pDG1730-yutB
pIS-comE-B	5′-ATCGGATCCTTTTGATGAGACACACGAGC-3′	pIS-comE
pIS-comE-H	5′-GATAAGCTTGTAGACGAGTATGTCATAC-3′	pIS-comE
T5-B1	5′-ATCGGATCCTCGAGAAATCATAAAAAATTT-3′	pIS-PT5
T5-H1	5′-GATAAGCTTGTGTGAAATTGTTATCCG-3′	pIS-PT5
comE-UTR-H3	5′-AGCTTTTCCCCTCCTTTGAGCTATTATCCTCTAAACCAT-3′	pIS-PT5-UTR
comE-UTR-H4	5′-AGCTATGGTTTAGAGGATAATAGCTCAAAGGAGGGGAAA-3′	pIS-PT5-UTR

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Plasmid construction.

Synthetic oligonucleotides were commercially prepared by the Tsukuba Oligo Service (Ibaraki, Japan). The plasmids and oligonucleotides used in this study are listed in Tables 1 and 2, respectively. To construct pDG1730-comN and pDG1730-yutB, PCR products produced by oligonucleotide pairs yrzD-op-Ba/yrzD-H and yutB-op-Ba/yutB-op-Hi, respectively, were digested by BamHI and HindIII and cloned into pDG1730 with the same restriction enzymes (15). To construct pPhl-yutB, a PCR product was prepared by using primers yutB-Sa and yutB-E, and the DNA fragment was digested with SalI and EcoRI and ligated into similarly digested plasmid pPhl-2 (36). For pMut-comEA, as we described previously, the fusion was constructed by cloning a PCR product generated by using oligonucleotides comE-U and comE-D into pMutinIII (35). Descriptions of the wrong oligonucleotides were given previously, and the correct oligonucleotides were comE-H and comE-Bg (Table 2). To construct pMut-comEA2, pMut-comEA3, pMut-comEA4, and pMut-comN, PCR products produced by using oligonucleotide pairs comE-B2/comE-H2, comE-B3/comE-H3, comE-B4/comE-H4, and yrzD-Mut-B/yrzD-Mut-H, respectively, were digested with BamHI and HindIII and cloned into pMutinIII using the same restriction enzymes (45). To construct pMut-comEA2a, pMut-comEA2b, and pMut-comEA21, PCR products produced by using oligonucleotide pairs comE-B2a/comE-H3, comE-B2b/comE-H3, and comE-B21/comE-H2, respectively, were digested with BamHI and HindIII and cloned into pMutinIII using the same restriction enzymes. To construct pIS-comE and pIS-PT5, PCR products were prepared from the total DNA of strain 168 and pQE8 (Qiagen) by using primer pairs pIS-comE-B/pIS-comE-H and T5-B1/T5-H1, respectively. The DNA fragments were digested with BamHI and HindIII and ligated into similarly digested plasmid pIS284 (44). The resultant plasmids were transformed into strain 168 after linearization by PstI digestion, which generated strains carrying comEA5-lacZ and P_T5-lacZ at amyE. To construct pIS-PT5-UTR, synthetic oligonucleotides comE-UTR-H3 and comE-UTR-H4 were hybridized, and then the resultant double-stranded DNA was cloned into the HindIII site of pIS-PT5. The direction of the DNA insert in the HindIII site was confirmed by PCR analysis. To construct pRB-comN, the cloned comN region of pDG1730-comN was retrieved by digestion of this plasmid with HindIII and BamHI, and the resultant comN fragment was cloned into pRB373 digested with the same enzymes (5). The PCR-amplified sequences cloned into all plasmids were confirmed. During this process we found a sequencing error in the comER ORF (accession number L15202 [14]) (GA and not CC at nucleotides 803 and 804).

Lac assay.

The β-galactosidase activities of lacZ fusions were determined as described previously (36).

RESULTS

Identification of YRZDc and YUTBd with decreased transformation frequencies.

Approximately 1,800 disruptants were constructed by the Japan Bacillus Functional Genomics Consortium, and we screened mutants with decreased transformation frequency. In the first round of screening, we selected mutants with a smaller number of transformants than the wild type (less than 5%). The second round of screening, to test the reproducibility of the decrease in transformability, was carried out by verifying the growth rates and abilities to form colonies on LB agar plates. As a result, we identified YRZDc and YUTBd in addition to the previously identified isolate SODd (a sodA disruptant [38]), in which the transformation frequency was greatly decreased (Table 3). The other disruptants identified in this screening will be described elsewhere. Next, we constructed backcrossed strains in which the gene disruptions were transformed into wild-type strains. The transformation frequencies of these backcrossed mutants were subsequently examined to eliminate any possible effects of a secondary mutation(s). As shown in Table 3, both of the gene disruptions in the backcrossed strains resulted in reduced transformation frequencies, although the transformation rate of the backcrossed yutB strain was somewhat higher than that of the original strain. This discrepancy might indicate that there was some unknown mutation that further reduced the transformation efficiency in the original YUTBd strain. The operon structures of the genes are shown in Fig. 1A. The disruptants with disruptions in the genes located downstream of the identified genes did not exhibit significant decreases in transformation frequency (Fig. 1A). Therefore, we concluded that the observed low transformability in these gene disruptants was not due to a polar effect.

TABLE 3.

Transformation frequencies in various mutants

Expt	Strain	Relevant genotype	No. of viable cells/ml (10⁶)^a	No. of transformed cells/ml (10)^a	Frequency (10⁻⁴)	Relative frequency (%)
Screening test (second round)	168	Wild type	314	2,400	0.76	100
	YRZDc	comN::Cm^r	114	0	0	0
	YUTBd	yutB::Em^r	172	14	0.008	1.1
Backcross expt and transformation test using newly constructed strain
Expt 1	168	Wild type	206	15,600	7.6	100
	YUTBd	yutB::Em^r	124	840	0.68	9
Expt 2	168	Wild type	374	35,100	9.4	100
	OAM304	yutB::Pm^r	203	2,930	1.4	15
Expt 3	168	Wild type	232	21,200	9.1	100
	YRZDc	comN::Cm^r	112	57	0.051	0.56
Complementation test	168	Wild type	193	19,300	10	100
	YRZDc	comN::Cm^r	162	72	0.043	0.43
	OAM434	comN::Cm^ramyE::comN	248	15,600	6.3	63
	OAM304	yutB::Pm^r	138	1,600	1.2	12
	OAM435	yutB::Pm^ramyE::yutB	178	19,800	11	110

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The numbers of colonies on LB agar plates containing serially diluted cultures were counted, and the results were calculated to the third significant digit.

FIG. 1. — Chromosomal locations of the *comN* (*yrzD*) and *yutB* genes and structure of the *comEA* operon. (A) The shaded and open boxes indicate the *comN* (*yrzD*) and *yutB* genes and the genes located peripherally, respectively. The numbers above and below the boxes indicate the relative transformation frequencies of the disruptants compared with the wild-type strain (expressed as percentages), which were determined by the screening analysis. (B) Schematic diagram of the *comE* operon structure. The standard and thin bent arrows indicate major and minor promoters, respectively. The open boxes indicate ORFs.

Next, we constructed a new yutB mutant that carried the phleomycin resistance gene via Campbell-type recombination using a pPhl-2-based plasmid (36). The yrzD mutant was generated by a gene replacement method using a chloramphenicol resistance gene, because the ORF is very small and use of this ORF has a tendency to result in a very low frequency of Campbell-type recombination. Thus, we did not construct a pPhl-2-mediated disruptant of yrzD. The transformation frequency of the new yutB mutant was less than that of the control strain (Table 3).

Next, the ORF with its own upstream region was introduced into the amyE locus of the disruptants. This resulted in complementation of the low transformability shown by both of the disruptants (Table 3). Hence, we concluded that disruption of yrzD and disruption of yutB resulted in severe and relatively compromised decreases in the transformation frequency, respectively (Table 3). yrzD encodes a 98-amino-acid protein that is conserved among the Bacillus-Listeria group, and this gene was renamed comN. The comN strain produced a slightly lower number of viable cells on an LB agar plate (Table 3), while its growth curve was similar to that of the 168 strain in an MC liquid culture (data not shown). Thus, comN disruption might result in a slight decrease in the ability to form colonies. The yutB gene product has high sequence similarity to lipoate synthetase. Moreover, the YUTBd strain showed a slight growth defect in liquid MC medium, and addition of 10 ng/ml lipoate rescued this growth defect (Fig. 2A). Thus, it is likely that the yutB gene encodes lipoate synthetase, although we did not examine whether the yutB gene product has lipoate synthetase activity, because this issue is not directly related to the regulation of competence development. We noted that both disruptants had normal cell morphology in liquid MC medium (data not shown).

FIG. 2. — Effects of addition of lipoic acid on growth and *comEA-lacZ* expression in the *yutB* strain. Cells were grown in MC medium without (left panels) or with (right panels) lipoic acid (10 ng/ml). (A) Growth of OAM438 (*comEA-lacZ*) (•) and OAM347 (*comEA-lacZ yutB*) (▵). Growth was monitored at hourly intervals by using a Klett spectrometer. Cell densities are expressed in arbitrary units. (B) Expression of *comEA-lacZ*. Cells were sampled hourly, and β-galactosidase activities are expressed in Miller units. The x axis indicates the growth time (in hours) relative to the end of vegetative growth (T0). The symbols are the same as those in panel A.

Epistatic analysis of comN and yutB.

We next examined whether the expression of srfA and/or comK was the primary target of the mutations. The expression of srfA in both of the disruptants was essentially not changed, indicating that neither the comN nor the yutB gene regulates srfA expression (data not shown). Next we examined whether disruption of both genes affected the expression of comK. Surprisingly, disruption of both of the genes did not significantly alter the expression of comK (Fig. 3). Therefore, we hypothesized that some of the late competence gene expression might be decreased in the disruptants. To address this issue, we examined the effects of both of the gene disruptions on late com gene expression. As shown in Fig. 3, in the comN and yutB disruptants, the expression of comE was almost eliminated, whereas the expression of comG, comF, and comC did not change significantly. However, a slight decrease in comK-lacZ expression was observed in the yutB strain, which led to slight decreases in late com gene expression. We observed that in both of the disruptants the expression of ywpH-lacZ was not affected (data not shown). It is possible that lipoate, but not the yutB gene product, is required for the expression of comE. Addition of lipoate (10 ng/ml) slightly enhanced the levels of expression of comEA-lacZ in both the wild-type and yutB backgrounds; however, the presence of lipoate did not compensate for the decreased expression of comEA-lacZ in the yutB strain compared to the expression in the 168 strain (Fig. 2B). These results strongly implied that YutB does not function in regulation of comE by synthesizing lipoic acid. On the basis of these results, we concluded that the decrease in comE expression should result in low transformation frequencies in the comN and yutB disruptants.

FIG. 3. — Identification of *comE*-specific regulation. Cells were grown in MC medium and sampled hourly. The β-galactosidase activities of the indicated fusion constructs are expressed in Miller units. The x axis indicates the growth time (in hours) relative to the end of vegetative growth (T0). *comF-lacZ* is a translational fusion, and the other constructs are transcriptional fusion products. •, wild type; ○, *comN*; ▵, *yutB*.

Effects of the comN and yutB mutations on comE promoter activity.

The comE operon has been reported to have an unusual structure; namely, the operon is composed of four ORFs, comEA, comEB, comEC, and comER (Fig. 1B). The comER ORF is on the antisense strand relative to the other ORFs and encodes a protein similar to pyrroline-5′-carboxylate reductases. The antisense strand of comER is also composed of an unusually long 5′-UTR for the mRNA encoding comEA, comEB, and comEC, because a major promoter for comEA-comEB-comEC is located at the 3′ end of the comER ORF (Fig. 1B) (16). To further examine the regulatory mechanism of the comN and yutB genes, we constructed a comE promoter fusion with lacZ at the amyE locus. The resultant transcriptional lacZ fusion product, comEA5-lacZ, carried the region from position −242 to position +3 relative to the transcription start site, which contains the ComK-binding site and the core promoter. The comEA5-lacZ fusion should be independent of the possible influence of a UTR because of the lack of the sequence and reflects the promoter activity directly. The expression of the fusion was significantly decreased in the yutB strains but not in the comN strains (Fig. 4). These results demonstrated that disruption of yutB decreased the activity of the comE promoter, while disruption of comN did not affect the promoter activity. This suggested that YutB is a transcriptional activator of comE and that ComN affects comE expression at a post-transcription initiation level. It should be noted that the observed effect of the yutB disruption on the promoter-fusion construct was less severe than the effect on the comEA-lacZ fusion, which carries the entire 5′-UTR (Fig. 3 and 4), suggesting that the yutB mutation might affect the 5′-UTR of comE and result in further decreases in its transcription. In Northern blot analyses using a comEB-specific probe, a band corresponding to a 4.5-kb mRNA (probably containing comEA, comEB, and comEC) was detected for the wild-type strain, although hybridization signals corresponding to heavily degraded mRNA were also detected (data not shown). In the comN strain, a similar amount and pattern of the hybridized signal corresponding to comE were observed, suggesting that there was no change in comE transcription in the comN mutant (data not shown). This is consistent with the results of the lacZ fusion analysis.

FIG. 4. — Expression of various *comEA-lacZ* fusions at the *amyE* or *comE* locus. (A) Schematic diagrams showing the 5′ and 3′ endpoints relative to the transcription start site of the *comEA* gene. *comEA5-lacZ* is a fusion at the ectopic *amyE* locus. The 3′ endpoints of the other fusion constructs, which were generated using Campbell-type recombination of the pMutin-based plasmids, are indicated. The small open boxes following the *lacZ* gene and the gray boxes following the *comEA* ORF indicate the Shine-Delgarno sequences of *spoVG* and *comEA*, respectively. The double slashes indicate the endpoint of the DNA sequence derived from pMutinIII DNA. The gray rectangles in each fusion indicate the DNA region cloned into the plasmid used for construction of the strain. The bent arrows indicate *comE* promoters. The bacterial cells were grown in MC medium, and their activities were assayed hourly from the end of vegetative growth to 4 h after the end of vegetative growth. The WT, *comN*, and *yutB* columns show the β-galactosidase activities of each fusion in the wild type, *comN*::Cm^r and *yutB*::Pm^r backgrounds, respectively. At least three independent assays were performed, and the averages of the peak values are shown. The standard deviations did not exceed 25%. ND, not determined. (B) Typical expression profiles of the fusions. The β-galactosidase activities of the indicated fusion constructs are expressed in Miller units. The x axis indicates the growth time (in hours) relative to the end of vegetative growth (T0). •, wild type; ○, *comN*; ▵, *yutB*.

5′-UTR deletion analysis reveals the post-transcription initiation control of comE by comN.

The results of lacZ fusion analyses suggested that the comN gene positively regulates comE expression at the post-transcription initiation level. Thus, we hypothesized that a cis element for post-transcription initiation control by comN is located in the 1-kb 5′-UTR of comE. To test this hypothesis, we constructed lacZ fusions with sequential deletions of the 5′-UTR, which were introduced into the comE locus by Campbell-type recombination. We measured the β-galactosidase activities of the resultant strains in the wild-type and comN backgrounds. The expression of comEA4-lacZ, comEA3-lacZ, comEA2b-lacZ, comEA2a-lacZ, and comEA21-lacZ, whose 3′ endpoints of the 5′-UTR are at positions +358, +558, +658, +758, and +823 relative to the transcription start site, respectively, was not affected by introduction of the comN mutation (Fig. 4). On the other hand, the expression of comEA2-lacZ and comEA-lacZ, whose 3′ endpoints are at positions +858 and +1177 relative to the transcription start site, respectively, was severely decreased by introduction of the comN mutation (Fig. 4). These results demonstrated that the cis element responsible for the post-transcription initiation control by comN is in the region from position +824 to position +858 of the 5′-UTR, which does not contain the Shine-Delgarno sequence of the comEA ORF. The fusions constructed are transcriptional lacZ fusions, and thus inhibition of the expression of lacZ occurs at the post-transcription initiation level in the cells carrying comEA-lacZ or comEA2-lacZ. The upstream cis element of comN probably would block expression of the gene located downstream from the cis element in the absence of ComN. We also noted that ComER should not be involved in this post-transcription initiation control. A promoter of the comER gene was in a distal region far from the ORF in the strain carrying comEA-lacZ, and the comER gene was disrupted in the other fusion constructs. These structural features probably lead to a loss of transcription of comER. The effects of the comN mutation, however, differed in these strains, suggesting that comER has no role in comEA regulation.

Effects of comN mutation and its cis element on a heterologous promoter.

To examine possible effects of the comN mutation and its cis element on a heterologous promoter, we fused the phage T5 promoter carrying the LacI operator sequence with promoterless lacZ (P_T5-lacZ) (6) and introduced it into the amyE locus of a strain carrying lacI. When 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a culture of the strain carrying this fusion, expression of lacZ resulted in accumulation of β-galactosidase (Fig. 5). As expected, introduction of the comN disruption did not affect P_T5-lacZ expression (Fig. 5A). Next, we introduced the 35-bp DNA region (positions +824 to +858) containing the cis element of the comN mutation between the transcription initiation site and the lacZ gene of the P_T5-lacZ fusion (Fig. 5C). The level of expression of the P_T5-UTR-lacZ fusion was about threefold lower than that of P_T5-lacZ, indicating that the introduced UTR sequence negatively affected the expression of the downstream lacZ gene. A similar inhibitory effect of this UTR was observed with the comE-lacZ fusions, as shown by a comparison of the activities of comEA21-lacZ and comEA2-lacZ in the wild-type background (Fig. 4A). A disruption of comN moderately decreased the expression of PT5-UTR-lacZ, suggesting that ComN positively regulates lacZ expression through possible antagonism of the inhibitory effect of UTR. To examine the possibility that the low levels of expression of PT5-UTR-lacZ would be compensated for by amplification of the copy number of the comN gene, we constructed pRB-comN by using multicopy plasmid pRB373 (5). Introduction of pRB-comN did not affect the expression of PT5-lacZ compared to the expression in the strain carrying pRB373 (Fig. 5B). As expected, introduction of pRB-comN into the strain carrying PT5-UTR-lacZ increased its expression by a factor of three, although the level of expression of lacZ was still lower than the level of expression of the PT5-lacZ fusion (Fig. 5B). On the basis of all of the data, we concluded that ComN functions through its cis element in the expression of lacZ driven by the heterologous PT5 promoter.

FIG. 5. — Effects of the ComN-UTR system on P_T5-*lacZ* expression. All strains were grown in LB liquid medium containing 1 mM IPTG. Three independent Lac assays were performed, and the β-galactosidase activities (averages ± the standard deviations) are indicated. (A) •, OAM550 (wild type, P_T5-*lacZ*); ○, OAM546 (*comN*, P_T5-*lacZ*); ▴, OAM547 (wild type, P_T5-UTR-*lacZ*); ▵, OAM548 (*comN*, P_T5-UTR-*lacZ*). (B) The culture contained kanamycin (10 μg/ml). •, OAM550 (P_T5-*lacZ*) carrying the vector pRB373; ○, OAM550 (P_T5-*lacZ*) carrying pRB-comN; ▴, OAM547 (P_T5-UTR-*lacZ*) carrying the vector pRB373; ▵, OAM547 (P_T5-UTR-*lacZ*) carrying pRB-comN. (C) Schematic diagram of the structure of P_T5-UTR-*lacZ* at the *amyE* locus. The bent arrow and the gray box indicate the P_T5 promoter and the 35-bp region of the UTR of *comE* inserted into P_T5-*lacZ*, respectively. The dashed lines indicate hypothetical inhibitory effects. The other regulatory mechanisms could also be determined based on the data (see Discussion).

DISCUSSION

We identified the comN gene as a regulatory factor in competence development that functions through the 5′-UTR of the comE operon. In addition, ComN and its cis element in the 5′-UTR are a positive regulatory mechanism in the expression of lacZ driven by the heterologous promoter. The mode of action of ComN is not known, although it appears to be mediated through a post-transcription initiation event. In bacteria, some genes have unusually long 5′-UTR sequences that play roles in mRNA processing or stability, antitermination, or translational control (4, 21, 40, 47, 48). The possible function of the identified cis element of the comE UTR could be ascribed to one or more of the regulatory modes mentioned above. This question remains to be resolved. It has been hypothesized that the effects of the 5′-UTR are due to the secondary structures of mRNA. In some cases, a trans-acting factor binds to the 5′-UTR and affects gene expression. In addition, the secondary structure of the 5′-UTR of mRNA can serve as a riboswitch to which some specific ligands bind and cause translational attenuation or inhibition (31). Thus, whether ComN binds to the 5′-UTR of the comE operon needs to be determined; if it does, whether ComN regulates comE expression directly also needs to be determined. Otherwise, it is likely that some unknown protein that binds to the cis element or a secondary structure formed by part of the 5′-UTR blocks some post-transcription initiation event and that the comN gene product rescues this blocking event. On the basis of all of the data, we could not determine whether ComN regulates comE directly or indirectly. We identified three possible secondary structures (short stem-loop structures) in the 5′-UTR sequence containing the putative cis element for ComN, although the importance of finding such structures is obscure (see Fig. S1 in the supplemental material). It should be noted that this type of short stem-loop structure could be bound by an RNA-binding protein in the case of a conserved family designated RsmA, a translation regulator (28). Based on genome database analysis, the comE operon and comN are conserved among the Bacillus group. Interestingly, in this group, the comER-comEA-comEB-comEC structure (i.e., an unusually long 5′-UTR) is also conserved. This suggests that there is a functional relationship between comN and the 5′-UTR of comE, as well as selective pressure for the presence of both comN and the 5′-UTR of comE. Based on our lacZ analysis, comN is not required for comE expression if the cis element in the 5′-UTR of comE has been deleted. These observations are consistent with the hypothesis concerning the selective pressure for the presence of both comN and the 5′-UTR of comE.

The yutB gene product should be lipoate synthetase, and lipoate itself is a cofactor of the pyruvate dehydrogenase complex. The genes encoding the E1β and E2 subunits of pyruvate dehydrogenase (pdhB and pdhC, respectively) are involved in sporulation (14). Thus, we were interested in whether the pdh genes were also involved in competence development. The pdhB disruptant did not grow in MC medium, which contains citrate, however, and thus we were able to examine only the pdhC disruptant. We found that this strain had a normal transformation frequency (unpublished results), indicating that the E2 subunit is not involved in competence development. These results support the notion that YutB does not regulate competence development through synthesis of lipoate. Bifunctionality of an enzyme that regulates gene expression has been reported previously (9). Thus, the yutB gene products might have such bifunctionality.

The post-ComK regulatory pathway has been described previously. For example, both the BdbC and BdbD thiol-disulfide oxidoreductases are required for disulfide bond formation in ComGC (32). In this case, however, the expression of bdbCD requires ComK, and thus the posttranscriptional regulation of ComGC was found to be indirectly controlled by ComK. In a comZ mutant, expression of comG was enhanced without any change in comK expression (35). However, expression of comZ is also under the control of ComK (35, 36). In contrast, in three independent DNA microarray analyses that were described previously, comN and yutB were not identified as ComK-dependent genes, indicating that their expression should not be dependent on ComK (2, 18, 37), and this was confirmed (unpublished results).

The identification of comE-specific regulation raises the possibility that in ComK-activated cells there is an additional transformation checkpoint. Further examination of the regulation of comN and yutB might reveal a previously unknown pathway for differentiation into a mature competent cell. In a preliminary study, we examined the expression patterns of both of these genes and found that expression was not restricted in either case to the competence medium. In addition, the expression levels were very low (less than 5 Miller units for comN and around 20 Miller units for yutB in MC medium) (unpublished results). It should also be noted that expression of yutB in MC medium resulted in onset of the early stationary phase. Further analysis of this newly identified regulation pathway will likely give us a better understanding of competence development.

Supplementary Material

[Supplemental material]

supp_191_3_949__index.html^{(978B, html)}

Acknowledgments

We thank A. I. Aronson and K. Kobayashi for kindly supplying the bacterial strains used in this study. We also thank M. Koyano, S. Saito, Y. Kijima, S. Kinoshita, K. Kasagawa, Y. Nishikawa, M. Fujimoto, D. Kondo, T. Ohsawa, S. Ozaki, Y. Watanabe, Y. Mastuba, M. Aoki, M. Hanazaki, M. Ohsawa, Y. Chiba, A. Fujiwara, T. Ogamino, and H. Ikeda for performing screening work and for their technical assistance.

This work was mainly supported by a Grant-in-Aid for Scientific Research on Priority Areas (C) “Genome Biology” to T.T. and by a Grant-in-Aid for Scientific Research (C) to M.O. from the Ministry of Education, Science and Sports and Culture of Japan. Additional support was provided by the Research and Study Program of the Tokai University Educational System General Research Organization (to M.O.).

Footnotes

^▿

Published ahead of print on 21 November 2008.

^†

Supplemental material for this article may be found at http://jb.asm.org/.

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[r1] 1.Albano, M., R. Breitling, and D. Dubnau. 1989. Nucleotide sequence and genetic organization of the Bacillus subtilis comG operon. J. Bacteriol. 1715386-5404. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Berka, R. M., J. Hahn, M. Albano, I. Draskovic, M. Persuh, X. Cui, A. Sloma, W. Widner, and D. Dubnau. 2002. Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK. Mol. Microbiol. 431331-1345. [DOI] [PubMed] [Google Scholar]

[r3] 3.Bongiorni, C., S. Ishikawa, S. Stephenson, N. Ogasawara, and M. Perego. 2005. Synergistic regulation of competence development in Bacillus subtilis by two Rap-Phr systems. J. Bacteriol. 1874353-4361. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Bricker, A. L., and J. G. Belasco. 1999. Importance of a 5′ stem-loop for longevity of papA mRNA in Escherichia coli. J. Bacteriol. 1813587-3590. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

The Bacillus subtilis Late Competence Operon comE Is Transcriptionally Regulated by yutB and under Post-Transcription Initiation Control by comN (yrzD) ^▿^†

Mitsuo Ogura

Teruo Tanaka

Abstract