Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response

Abstract

Horizontal gene transfer contributes to the evolution of bacterial species. Mobile genetic elements play an important role in horizontal gene transfer, and characterization of the regulation of these elements should provide insight into conditions that influence bacterial evolution. We characterized a mobile genetic element, ICEBs1, in the Gram-positive bacterium Bacillus subtilis and found that it is a functional integrative and conjugative element (ICE) capable of transferring to Bacillus and Listeria species. We identified two conditions that promote ICEBs1 transfer: conditions that induce the global DNA damage response and crowding by potential recipients that lack ICEBs1. Transfer of ICEBs1 into cells that already contain the element is inhibited by an intercellular signaling peptide encoded by ICEBs1. The dual regulation of ICEBs1 allows for passive propagation in the host cell until either the potential mating partners lacking ICEBs1 are present or the host cell is in distress.

Horizontal gene transfer and mobile genetic elements play a significant role in bacterial evolution (1-4). Conjugative transposons (5, 6), also known as integrative and conjugative elements (ICEs) (4, 7), are mobile genetic elements that are normally integrated into the chromosome. They can excise and transfer to recipients through conjugation (mating) and integrate into the chromosome of the recipient (5, 6). ICEs encode proteins required for conjugal transfer and can also encode proteins involved in resistance to antibiotics (5, 6), metabolism of alternative carbon sources (4, 8), symbiosis (9), and other processes (10). ICEs and putative ICEs have been found in many bacteria (10) and are important agents of horizontal gene transfer because they are capable of moving themselves and other DNA to recipients (6, 11-13).

Mechanisms that regulate transfer have been determined for several ICEs. In some cases, an antibiotic induces transfer of an element that encodes resistance to that antibiotic (5, 6, 14). Transfer of the Streptomyces ICE pSAM2 is inhibited by the presence of a pSAM2-encoded protein in the recipient (15). Recently, it was shown that the DNA damage response stimulates transfer of SXT, an ICE from Vibrio cholerae (14).

We characterized a 20-kb ICE, ICEBs1 (16), in Bacillus subtilis and found that ICEBs1 excision and transfer is regulated by a secreted peptide encoded by ICEBs1.

Many Gram-positive bacteria use secreted signaling peptides to coordinate physiological processes with population density, often called quorum sensing (17). In B. subtilis, several secreted peptides contribute to quorum sensing, including Phr peptides encoded by phr genes (reviewed in ref. 18). It has been suggested that Phr peptides act as autocrine signals and not in cell-cell signaling (reviewed in ref. 19), although this is clearly not true for all Phr peptides (20, 21). Nonetheless, all characterized Phr peptides have a common mechanism of action. After secretion and extracellular accumulation, Phr pentapeptides are imported through the oligopeptide permease (Opp); once inside the cell, Phr peptides directly inhibit the activities of intracellular regulators, known as Rap proteins (20-24) (Fig. 1). The characterized Rap proteins directly (24, 25) or indirectly (23, 26) inhibit the activities of transcription factors that regulate sporulation, competence development, and production of degradative enzymes and antibiotics (20, 22-24).

Fig. 1.

Phr peptide signaling in B. subtilis. rap and phr genes are transcribed and translated (A); pre-Phr peptides are secreted and processed (B); mature Phr peptides are transported into the cell by the Opp (C); once inside the cell, Phr peptides inhibit the activities of regulators known as Rap proteins (D); each characterized Rap protein inhibits the activity of a transcription factor, either directly or indirectly (E); and inhibition of transcription factors lead to cellular responses (F).

RapI and PhrI are encoded by ICEBs1. We found that RapI activates ICEBs1 gene expression, excision, and transfer and that the PhrI peptide antagonizes the activity of RapI. Furthermore, expression of rapI and phrI is stimulated by conditions of low nutrient availability and high cell density. This combined regulation activates ICEBs1 excision and transfer when host cells are crowded by potential recipients that lack ICEBs1 and do not produce the PhrI peptide.

In addition, we observed that the global DNA damage (SOS) response activates ICEBs1 excision and transfer, independently of rapI and phrI. Therefore, at least two conditions promote ICEBs1 excision and transfer: the presence of a high concentration of cells lacking ICEBs1 and host cell distress. In the absence of these conditions, ICEBs1 is propagated by the host through vertical gene transfer to progeny cells.

Materials and Methods

Media. Cells were grown at 37°C, with agitation in LB medium (27), defined minimal medium (28) (supplemented with required amino acids when necessary), Schaeffer's nutrient broth sporulation medium (29), or brain heart infusion medium (29), as indicated. Antibiotics and other chemicals were used at the following concentrations: ampicillin (100 μg/ml), chloramphenicol (5 μg/ml), kanamycin (5 μg/ml), spectinomycin (100 μg/ml), streptomycin (100 μg/ml), erythromycin (0.5 μg/ml), and lincomycin (12.5 μg/ml) together, to select for macrolide-lincosamide-streptogramin B resistance, and isopropyl-β-d-thiogalactopyranoside (IPTG) (1 mM, Sigma) and mitomycin C (MMC) (1 μg/ml, Sigma).

Strains and Alleles. Strains used in this study are listed in Table 3, which is published as supporting information on the PNAS web site. The Escherichia coli strain used for cloning is an MC1061 derivative carrying F′(lacI^q) lacZM15 Tn10 (tet). Standard techniques were used for cloning and strain construction (27, 29).

For overexpression in B. subtilis, rapI, phrI, and rapI phrI were cloned downstream of the IPTG-inducible promoters Pspank(hy) (30) or Pspank (28), both generous gifts from D. Rudner (Harvard Medical School, Boston), and integrated into the amyE locus by homologous recombination. Pspank and Pspank(hy) (with no inserts) were also integrated into amyE.

The rapI-lacZ promoter fusion was generated by cloning the DNA from 329 to 12 bp upstream of the rapI ORF upstream of the promoterless lacZ in the vector pDG793 (31), followed by integration into the thrC locus by homologous recombination.

Isolation of spontaneous streptomycin-resistant mutants and construction of the following alleles is described in Supporting Methods, which is published as supporting information on the PNAS web site: ICEBs1::kan, an insertion of a kanamycin-resistance gene between the 3′ end of yddM and attachment (att) site attR; ICEBs1⁰, a complete loss of ICEBs1 that leaves the chromosomal att site intact; and Δ(ICEBs1)206::cat, a deletion of the entire ICE, including attR, and insertion of a chloramphenicol-resistance gene. Null mutations included Δ(rapI phrI)342::kan, Δint205::cat, and ΔimmR208::cat. S. Branda and R. Kolter generously provided ΔphrI173::erm and Δ(rapI phrI)260::erm.

comK::spc and comK::cat (32), ΔabrB::cat (33), recA260 (34, 35), and opp::cat [opp::Tn917lac::pTV21Δ2cat (opp = spo0K)] (36), were described previously.

DNA Microarrays. Cells were harvested, and total RNA was prepared as described in ref. 30. RNA from each sample was reverse-transcribed and labeled with Cy3 or Cy5. Labeled samples were combined and purified with Qiagen PCR purification columns and hybridized to microarrays containing PCR products of virtually all of the B. subtilis ORFs (30). Similar hybridization experiments were performed by using microarrays containing a unique DNA oligonucleotide for each B. subtilis ORF. Additional details are described in Supporting Methods.

Arrays were scanned and analyzed with the program genepix 3.0 (Axon Instruments, Union City, CA). Cy3 and Cy5 signals for each spot were normalized to the total Cy3 and Cy5 signals of the array and were obtained for each spot that had a signal above background for 50% of pixels. Iterative outlier analysis (30, 37) was used to identify spots (genes) whose experimental mean ratio was >2.5 SDs away from the mean ratio of the population of genes in the third iteration of the calculation (outlier cutoff). The probability that the mean ratios of these outliers were greater than the outlier cutoff was calculated by using the normal distribution function for each spot; those genes with ≥95% probability were considered significantly changed. The mean ratio for a set of triplicate experiments is reported.

Excision Assays. DNA was extracted by using the Qiagen DNEasy tissue kit (protocol for Gram-positive bacteria with RNase A treatment). PCR with the primer pair oJMA93 and oJMA100 detected the chromosomal junction formed after ICEBs1 excision. PCR with the primer pair oJMA95 and oJMA97 detected the excised ICEBs1 circle. Primer sequences, PCR conditions, and cycling parameters are described in Supporting Methods. Products were visualized on 2% agarose gels stained with ethidium bromide. PCR was performed on at least two independent biological replicates. Representative results are shown.

For linear-range (quantitative) PCR, known concentrations of DNA were diluted serially, and regions were amplified by using the indicated primer pairs. Products were visualized on 2% agarose gels stained with ethidium bromide and quantified by using the ChemiImager gel documentation system (Alpha Innotech, San Leandro, CA). Reactions were deemed in the linear range when three 2-fold serial dilutions of input DNA produced linearly decreasing amounts of PCR product.

The relative increase in excision is reported for circular intermediate PCR products. Fold-increase was determined by calculating the amount of PCR product of the ICEBs1 circle in each experimental sample, compared with the amount of ICEBs1 circle PCR product from the control sample for each experiment. These fold-increases were normalized to the amount of PCR product from cotF for each sample. cotF, a chromosomal site unaffected by ICEBs1 excision, was amplified with primers oLIN93 and oLIN94 (38). The fold-increase is reported as the mean (±SEM) from at least two independent experiments.

In experiments with mixed cultures, an additional normalization was done to take into account only the cells capable of excision of ICEBs1. PCR was also done with the primer pair oJMA177 and oJMA178 that amplifies DNA [amyE::Pspank(hy)] unique to the population of cells capable of excision. The amount of this product in the experimental sample was compared with the amount of this product in the control to determine the number of cells in the experimental sample capable of excision. All cells in the control were capable of excision and contained amyE::Pspank(hy).

Mating Experiments. Donors and recipients were grown in LB (for matings with Bacillus) or brain heart infusion (BHI) medium (for matings with Listeria) when assaying transfer from cells overexpressing rapI or in defined minimal medium when assaying transfer from cells treated with MMC. ICEBs1 excision in donor cells was induced either by overexpression of rapI [Pspank(hy)-rapI Δ(rapI phrI), strain JMA168] or addition of MMC [Δ(rapI phrI), strain IRN342]. At 1 h after induction, equal volumes of donor and recipient cultures were mixed and filtered onto a sterile nitrocellulose filter (0.2 μm pore size, Nalgene), placed on LB or BHI agar plates, and incubated at 37°C for ≈3 h. Cells were removed from filters by washing with 5 ml of Spizizen minimal salts (29). Transconjugants were isolated by selecting for antibiotic resistance unique to the recipient and the kanamycin resistance in ICEBs1. Donor and recipient numbers were also determined by selective plating. Concentrated, unmixed donor and recipient cultures spread on the double-antibiotic agar did not give rise to spontaneous antibiotic-resistant mutants. Transfer of DNA to the donor through transformation was not observed.

Mating frequencies were calculated by dividing the number of transconjugants by the number of donor cells, except in the case of donor cells treated with MMC, where mating frequencies were calculated relative to recipients. The reported transfer frequencies are the mean (±SEM) of at least two independent biological replicates.

β-Galactosidase Assays. β-Galactosidase-specific activity of a rap-I-lacZ fusion was assayed throughout growth of wild-type and ΔabrB cultures in sporulation media, as described in ref. 39.

Results and Discussion

Identification of a Mobile Genetic Element Regulated by Peptide Signaling. B. subtilis encodes seven phr genes (40), each located in an operon with a rap gene. To identify biological processes regulated by the uncharacterized rapI-phrI operon, we used whole-genome DNA microarrays to monitor changes in mRNA levels caused by overexpression of rapI from an IPTG-inducible promoter [Pspank(hy)-rapI].

In two types of microarray experiments, overproduction of RapI caused mRNA levels of 18 genes to increase ≥4-fold (Fig. 2; and see Table 4). All 18 genes cluster around rapI and phrI and are in the 20-kb ICEBs1 element (Fig. 2) previously identified by comparative sequence analysis (16). ICEBs1 is flanked by 60-bp direct repeats, the likely att sites. One of the potential att sites is in the 3′ end of a tRNA gene, a common integration site for mobile elements (41). ICEBs1 contains int (previously ydcL) (16), encoding a putative λ-like integrase, immR (previously ydcN), encoding a putative bacteriophage-like immunity repressor with 50% amino acid similarity to the repressor of B. subtilis phage φ105 (42, 43), and seven genes similar to genes from other ICEs (16). Our results demonstrate that RapI activates ICEBs1 gene expression. This activation is most likely by directly or indirectly inhibiting the activity of the putative immunity repressor ImmR (J.M.A. and A.D.G., unpublished data). Furthermore, activation of ICEBs1 gene expression is specific to overexpression of rapI, because overproduction of other B. subtilis Rap proteins did not stimulate ICEBs1 gene expression (J.M.A. and A.D.G., unpublished results).

Fig. 2.

Overexpression of rapI activates expression of genes in ICEBs1. The diagram shows the organization of ICEBs1, which contains at least 24 ORFs. The name of each gene is indicated above its respective arrow. Black boxes at the left and right ends indicate the att sites attL and attR. attL of ICEBs1 is in the 3′ end of a leucyl-tRNA gene (trnS-leu2). The black arrow indicates int, encoding the putative integrase. The hatched arrow indicates immR, encoding the putative immunity repressor. Shaded arrows indicate genes similar to those found in other ICEs (16). The numbers below the cartoon of ICEBs1 indicate the mean fold-increase in mRNA levels in cells overexpressing rapI. Pspank(hy)-rapI (JMA28) cells were grown for at least four generations to midexponential phase in minimal medium. IPTG was added to half of the cultures to induce rapI expression. Samples were collected 30 min later from induced and uninduced cultures. RNA was isolated, labeled, and hybridized, and genes that changed significantly upon overproduction of RapI were identified, as described in Materials and Methods. Expression of the three genes at the left end did not change significantly nor did the expression of almost all chromosomal genes. Experimental details and additional microarray results are in Table 4 and Supporting Text, which are published as supporting information on the PNAS web site.

ICEBs1 Excises and Transfers. Before conjugal transfer, an ICE excises from the chromosome, forming a circular intermediate and a repaired chromosomal junction (5). We used a PCR-based assay to detect products formed upon ICEBs1 excision (Fig. 3A). We detected a low level of circular ICEBs1 intermediates and repaired chromosomal junctions in control cells of B. subtilis (Fig. 3B), indicating that excision occurs at a low level in this population of cells. Overexpression of rapI greatly stimulated ICEBs1 excision (Fig. 3B). Because expression of the putative integrase is not activated by rapI overexpression, RapI likely stimulates excision by activating expression of an accessory protein required for excision. Many integrase proteins require accessory proteins for excision (44).

Fig. 3.

Excision of ICEBs1. (A) PCR assay for determining excision of ICEBs1. Primers a and d (oJMA93 and oJMA100) anneal to sequences surrounding ICEBs1 and amplify the repaired chromosomal junction formed upon excision. Primers b and c (oJMA95 and oJMA97) anneal to sequences inside ICEBs1 and amplify the circular intermediate generated upon excision. (B) Overproduction of RapI and treatment with MMC induce ICEBs1 excision. Cells were grown to midexponential phase in minimal medium. Samples were collected 1 h after treatment with IPTG (to induce rapI overexpression) or MMC (to cause DNA damage and induce the SOS response). 100 ng of template DNA was used to amplify the indicated products. Shown are: lane 1, control cells [Pspank(hy), JMA35]; lane 2, Pspank(hy)-rapI (JMA28); lane 3, wild-type cells (JH642), untreated; and lane 4, wild-type cells treated with MMC. Induction of ICEBs1 excision by MMC was recA-dependent (data not shown). (C) PhrI pentapeptide inhibits ICEBs1 excision. Cells [Pspank-rapI Δ(rapI phrI); JMA342] were grown to midexponential phase in minimal medium. Where indicated, the synthetic PhrI pentapeptide (DRVGA) in potassium phosphate buffer, pH 7 (Genemed Synthesis, South San Francisco, CA) was added to cultures at 100 nM and 1 μM. Buffer was added to the control cultures; all cultures had a final buffer concentration of 1 mM. Ten minutes later, IPTG was added to induce RapI overproduction. Samples were collected 1 h after IPTG addition, and linear-range PCR was performed as described (Materials and Methods). Pspank-rapI [rather than Pspank(hy)-rapI] was used, because transcription from Pspank is better repressed in the absence of inducer. Open bar, uninduced cells, defined as 1; black bar, overproduction of RapI; shaded bar, overproduction of RapI, in 100 nM PhrI pentapeptide; hatched bar, overproduction of RapI, in 1 μM PhrI pentapeptide. (D) Opp is required for phrI to inhibit excision. Cells were grown to midexponential phase in minimal medium. Samples were collected 1 h after addition of IPTG and analyzed by linear-range PCR. Open bar, overexpression of rapI alone [Pspank(hy)-rapI Δ(rapI phrI), JMA168], defined as 100%; black bar, overexpression of rapI and phrI [Pspank(hy)-(rapI phrI) Δ(rapI phrI), JMA186]; shaded bar, overexpression of rapI in an opp-null mutant [Pspank(hy)-rapI Δ(rapI phrI) Δopp, CAL51]; hatched bar, overexpression of rapI and phrI in an opp-null mutant [Pspank(hy)-(rapI phrI) Δ(rapI phrI) Δopp, CAL52]. (E) Excision of ICEBs1 increases in a phrI-null mutant. Cells were grown in nutrient broth sporulation medium. Samples were collected from cells ≈2 h after the entry into stationary phase, and relative excision of ICEBs1 was determined by linear-range PCR. Open bar, wild-type (NCIB3610), defined as 1; black bar, ΔphrI (SSB173); shaded bar, Δ(rapI phrI) (SSB260); hatched bar, ΔphrI Pspank(hy)-phrI (JMA298). -/c indicates complementation of ΔphrI mutation.

rapI overexpression also stimulated ICEBs1 transfer to recipients. To assay transfer from donor cells, we replaced rapI and phrI with an antibiotic-resistance marker. Deletion of rapI and phrI had minimal effects on the excision of ICEBs1 in wild-type cells (Fig. 3E) and in cells overexpressing rapI (data not shown). We assayed transfer of ICEBs1 on a solid surface (filter mating) by mixing donor cells [Pspank(hy)-rapI Δ(rapI phrI)::kan], in which rapI overexpression had been induced for 1 h, with an equal number of recipient B. subtilis cells that lacked ICEBs1 (ICEBs1⁰).

ICEBs1 transferred at an average frequency of ≈1 × 10^-2 transconjugants (recipients that received ICEBs1) per donor (Table 1). Transfer into recipients that contained ICEBs1 occurred with ≈50-fold lower frequency (Table 1), indicating that ICEBs1 encodes at least one mechanism that inhibits acquisition of a second element. Acquisition of ICEBs1 by recipients was not due to natural transformation, because the recipients were comK mutants incapable of transformation (32). Transfer of ICEBs1 from nonactivated donor cells [Δ(rapI phrI)::kan, IRN342] was not detected under these conditions (<2 × 10^-8 transconjugants per donor).

Table 1. Frequency of ICEBs1 mating into recipients.

Recipient	Mating frequency*
B. subtilis ICEBs10 (CAL89)	1 × 10−2 ± 3 × 10−3
B. subtilis ICEBs1+ (CAL88)	2 × 10−4 ± 1 × 10−4
B. anthracis (UM44-1C9)	6 × 10−3 ± 5 × 10−3
B. licheniformis (REM42)	2 × 10−4 ± 5 × 10−6
L. monocytogenes (10403S)	8 × 10−6 ± 6 × 10−6

^*Mating was assayed 1 h after induction of rapl overexpression from donor cells (Pspank(hy)-rapl δ(rapl phrl)::kan, JMA 168). Mating frequency is the number of transconjugants per donor (±SEM).

Transfer of ICEBs1 into Bacillus and Listeria Recipients. The putative bacterial chromosomal att site of ICEBs1 is conserved (≥52 of 60 base pairs identical) in Bacillus, Listeria, and Staphylococcus species (see Fig. 5, which is published as supporting information on the PNAS web site). We assayed transfer of ICEBs1 from B. subtilis donor cells overexpressing rapI into Bacillus anthracis, Bacillus licheniformis, and Listeria monocytogenes and found that ICEBs1 mated into all three species (Table 1). The efficient transfer of ICEBs1 into Bacillus and Listeria species, and, potentially, Staphylococcus species (not tested), indicates that ICEBs1 may be a useful tool to facilitate genetic manipulation of these organisms.

Inhibition of ICEBs1 Excision by the PhrI Peptide. Because the activities of the characterized Rap proteins are inhibited by their cognate Phr peptides and rapI overexpression activates ICEBs1 excision and transfer, we investigated whether PhrI peptide signaling inhibits ICEBs1 excision and transfer. Excision of ICEBs1 in cells overexpressing rapI was inhibited by the addition of synthetic PhrI peptide (Fig. 3C). The active PhrI peptide, the five C-terminal amino acids of the 38-aa precursor protein, was predicted based on its similarity to characterized Phr peptides (18, 19). The addition of 1 μM synthetic PhrI peptide inhibited RapI-dependent excision of ICEBs1 ≈20-fold, and addition of 100 nM PhrI peptide inhibited excision ≈3-fold (Fig. 3C). These concentrations of peptide are similar to the biologically active concentrations of other Phr peptides (21, 23, 45). These results demonstrate that the PhrI pentapeptide inhibits RapI-dependent activation of ICEBs1 excision.

Excision of ICEBs1 in cells overexpressing rapI was inhibited ≈50-fold by cooverexpression of phrI (Fig. 3D). This inhibition depended on the presence of the opp, a transporter required for uptake of Phr peptides (20, 22, 23). Excision occurred at similar levels in opp^- cells cooverexpressing rapI and phrI and in opp⁺ cells overexpressing rapI alone (Fig. 3D). These data provide further evidence that the secreted PhrI peptide is imported through the Opp and inhibits RapI-dependent activation of ICEBs1 excision.

PhrI also inhibits ICEBs1 excision when rapI is expressed from its native promoter. Deletion of the gene encoding PhrI (ΔphrI), in otherwise wild-type cells, activated ICEBs1 excision >5,000-fold, relative to wild-type cells (Fig. 3E). This increase required RapI; excision in Δ(rapI phrI) cells was similar to wild-type (Fig. 3E). Ectopic expression of phrI complemented the ΔphrI phenotype, reducing ICEBs1 excision back to a low level (Fig. 3E), indicating that increased excision in the ΔphrI mutant was due to loss of phrI and not to effects on neighboring genes.

Regulation of ICEBs1 Excision and Transfer by Intercellular Signaling. The preceding results indicated that PhrI peptide signaling inhibited ICEBs1 excision but did not indicate whether the PhrI peptide acts as an intercellular signaling peptide. If the PhrI peptide acts as an intercellular signaling peptide, then RapI-dependent activation of ICEBs1 excision and transfer should be inhibited when the concentration of PhrI peptide produced by the population of cells is high, as when the majority of cells in the population contain ICEBs1 and produce PhrI. However, when the concentration of PhrI peptide is low, as when the majority of cells in the population lack ICEBs1 and do not produce the PhrI peptide, then RapI-dependent activation of ICEBs1, excision, and transfer should occur. ICEBs1 could use this mechanism to inhibit excision and transfer when surrounded by cells that already contain ICEBs1.

To test this model, we monitored excision in a minority population of ICEBs1⁺ cells when they were grown together with a majority of ICEBs1-containing cells that either produced PhrI (phrI⁺) or did not produce PhrI (ΔphrI) (Fig. 4A). In these mixed cultures, only the minority ICEBs1⁺ cells were capable of excision, because cells in the majority lacked integrase (Δint), which is required for ICEBs1 excision (C.A.L. and A.D.G., unpublished results).

Fig. 4.

Excision is inhibited in the presence of PhrI⁺ cells. (A) Outline of mixing experiments. A minority population (≈4% of total) of cells capable of ICEBs1 excision and transfer (Excision⁺ PhrI⁺) was mixed with a majority population (≈96% of total) of cells incapable of ICEBs1 excision and transfer that either did (Excision^- PhrI⁺) or did not (Excision^- PhrI^-) encode PhrI. (B) Excision of ICEBs1 in cells grown in mixed culture with a majority of ICEBs1 Excision^- PhrI⁺ (JMA205, open bars) or ICEBs1 Excision^- PhrI^- (JMA304, black bars) cells was measured during exponential growth and ≈2 h after the entry into stationary phase. Cells were grown separately in nutrient broth sporulation medium to midexponential phase. Cells were diluted into fresh medium at a ratio of ≈1 minority cell [JMA35, Pspank(hy)] to 24 majority cells [JMA205 (Δint) or JMA304 (Δint ΔphrI)] to a total OD₆₀₀ of ≈0.015-0.03 and were cocultured throughout growth. Samples were collected during midexponential growth (OD₆₀₀ ≈0.2) and ≈2 h after cells entered stationary phase and were used for linear-range PCR assays. In addition to the circular intermediate and chromosomal control (cotF) primer pairs (see Materials and Methods), the primer pair oJMA177 and oJMA178 was used in linear-range PCR assays to amplify a sequence specific to Pspank(hy), which is present in only the minority JMA35 cells. The amount of circular intermediate product from each experimental sample was normalized to the amount of Pspank(hy) and cotF products in that sample. This ratio was normalized to the amount of circular intermediate product in an unmixed Pspank(hy) culture (JMA35), also normalized to the amount of Pspank(hy) and cotF products, at each time point (defined as 1, data not shown) to give the relative increase in excision.

During midexponential growth, ICEBs1 excision was low, whether minority ICEBs1⁺ cells were grown with excess phrI⁺ or ΔphrI cells (Fig. 4B). However, ≈2 h after the cells entered stationary phase, ICEBs1 excision was stimulated >40-fold in the ICEBs1⁺ cells mixed with ΔphrI cells, relative to ICEBs1⁺ cells mixed with phrI⁺ cells (Fig. 4B). We observed a similar increase in excision when ICEBs1⁺ cells were mixed with cells lacking ICEBs1 (data not shown).

These results indicate that the PhrI peptide acts as an intercellular signaling peptide that inhibits ICEBs1 excision when cells are crowded by cells that contain ICEBs1 and produce the PhrI peptide. Furthermore, ICEBs1 excision is inhibited in exponential growth, irrespective of whether cells in the majority population contain phrI, indicating that an additional mechanism inhibits ICEBs1 excision and transfer. AbrB is a transitionstate regulator that represses transcription of several B. subtilis genes during exponential phase and is inactive under conditions of nutrient limitation and high cell density (reviewed in ref. 46). We found that transcription of rapI, measured with a rapI-lacZ promoter fusion, increased ≈5-fold in an abrB mutant (CAL26), relative to wild-type cells (CAL15), indicating that AbrB represses rapI transcription, either directly or indirectly. Consistent with this model, we also found that ICEBs1 excision increased in ΔabrB cells, relative to wild-type cells; this effect was much larger in exponential phase than in stationary phase (see Fig. 6, which is published as supporting information on the PNAS web site).

Taken together, these observations indicate that at least two mechanisms regulate RapI-dependent activation of ICEBs1 excision. When nutrients are abundant and cell density is low, AbrB represses rapI transcription, preventing RapI-dependent activation of ICEBs1 excision. As cells enter stationary phase, rapI transcription is derepressed and RapI can activate excision but only when the concentration of PhrI peptide is too low to inhibit RapI.

As expected, transfer of ICEBs1 was also inhibited when potential donors were surrounded by cells that produced the PhrI peptide. We introduced an antibiotic-resistance cassette into ICEBs1 between the last gene of the element (yddM) and the attachment site attR. This insertion did not have a significant effect on mating frequency; donor cells overexpressing rapI that contained this insertion (JMA448) or an antibiotic insertion in rapI and phrI (JMA168) mated at similar frequencies (data not shown). We tested transfer of ICEBs1 from a minority population (ICEBs1::kan) into cells in the majority population that either did (phrI⁺ Δint comK) or did not (ΔphrI Δint comK) produce PhrI. ICEBs1 transfer in the mixed cultures, measured 2 h after cells entered stationary phase, was >100-fold higher into recipients that lacked phrI than into cells that contained phrI (Table 2). This stimulation depended on RapI; it did not occur when the donor cells lacked rapI and phrI (Table 2).

Table 2. Transfer of ICEBs1 is inhibited if the surrounding cells are phrl⁺.

	Recipient
Donor	ICEBs1+ Excision− Phrl+	ICEBs1+ Excision− Phrl−
rapl+phrl+	1.0 × 10−5 ± 4.0 × 10−6	3.0 × 10−3 ± 1.0 × 10−3
Δ(raplphrl)	1.0 × 10−5 ± 4.0 × 10−6	5.0 × 10−6 ± 2.0 × 10−6

A minority population of ICEBs1-containing cells (potential donors), containing an antibiotic-resistance gene in ICEBs1 (ICEBs1::kan rapl⁺ phrl⁺, JMA384) was grown in mixed culture with a majority population of ICEBs1-containing cells (potential recipients) that were incapable of excision, defective in competence development; and either phrl⁺ (phrl⁺ Δint comK, JMA381) or phrl⁻ (Δphrl Δint comK, JMA306), as described in Fig. 4. To show dependence on rapl in the donor, a similar experiment was done with potential donors lacking rapl and phrl[Δ(rapl phrl)::kan, IRN342]. Strains were first grown separately in nutrient broth sporulation medium to midexponential phase. Cells were then diluted into fresh medium at a calculated ratio of ≈1 potential donor to 24 potential recipients (total OD₆₀₀ ≈0.015-0.03) and were grown in coculture until ≈2 h after entry into stationary phase. A 5-ml aliquot of each coculture was removed, mixed with 7.5 ml of fresh medium, filtered, and incubated on sporulation medium agar for ≈3 h. Filters were washed, and samples were plated selectively, as described in Materials and Methods. The mean number of transconjugants per donor cell (±SEM) for at least two independent experiments is reported. ICEBs1 transfer occurred much more efficiently under these mating conditions than under the conditions described in Table 1 (see Table 5, which is published as supporting information on the PNAS web site, and Supporting Text.

Taken together, the results of the excision and mating experiments indicate that ICEBs1 excision and transfer is more active when cells are crowded by potential mating partners that do not produce the PhrI peptide. Excision and transfer is limited to conditions that are likely to correlate with cell crowding, starvation, and high cell density, through the growth-phase-dependent regulation of rapI transcription. In this way, ICEBs1 uses intercellular peptide signaling to coordinate excision and mating with conditions that favor its productive dissemination to recipients lacking ICEBs1.

Activation of ICEBs1 Excision and Transfer by the SOS Response. Previous analysis of mRNA levels using DNA microarrays indicated that genes in ICEBs1 are activated by a variety of conditions that induce the SOS response (A. Goranov, E. Kuester-Schoeck, R. Britton, and A.D.G., unpublished results). Treatment of wild-type cells with MMC, a DNA-damaging agent that induces the SOS response in B. subtilis (47), stimulated ICEBs1 excision (Fig. 3B). Increased gene expression and excision in response to MMC depended on recA, which is required for the SOS response (47), and was independent of rapI and phrI (data not shown).

Mating frequency also increased when potential donor cells [Δ(rapI phrI)::kan, IRN342] were treated with MMC. The mean mating frequency was 2 × 10^-4± 8 × 10^-5 transconjugants per ICEBs1⁰ recipient (CAL89). Mating was undetectable from untreated cells under these conditions (<2 × 10^-8 transconjugants per recipient). Mating frequency was determined relative to recipients, because MMC treatment reduced the viability of donors. Induction of ICEBs1 excision and transfer by the SOS response may be an attempt by the element to escape the distressed cell for a viable host.

Conserved Signals Regulate Dissemination of Mobile Genetic Elements. We determined that ICEBs1 gene expression, excision, and transfer are inhibited by a self-encoded peptide and activated by the SOS response. Intercellular signaling also regulates transfer of some conjugative plasmids. Two well studied examples are transfer of the Ti plasmid in Agrobacterium tumafaciens (reviewed in ref. 48) and transfer of pheromone-inducible plasmids in Enterococcus faecalis (reviewed in refs. 49 and 50).

Ti plasmid transfer is stimulated by the presence of cells that contain the plasmid; this stimulation depends on the plasmid-encoded signal synthetase TraI, which synthesizes 3-oxo-8 homoserine lactone, and the plasmid-encoded regulatory protein TraR (48). In contrast, transfer of ICEBs1 is inhibited by the presence of cells that contain the element.

In Enterococcus faecalis, several mating pheromones (peptides) are encoded in the chromosome. Each pheromone stimulates transfer of a specific conjugal plasmid, and production of these pheromones by cells lacking specific plasmids stimulates transfer of those plasmids from donors (49, 50). Plasmid-containing cells also produce unique plasmid-encoded peptides that inhibit plasmid transfer to potential recipients that already contain the plasmid (49, 50).

Although peptides produced by Enterococcus faecalis pheromone-responsive plasmids and ICEBs1 both inhibit transfer, the regulatory mechanisms are different. With Enterococcus faecalis plasmids, specific peptide signals produced by recipients trigger transfer from donor cells. ICEBs1 transfer is stimulated by conditions (low nutrient availability and high cell density) likely to correlate with a high number of potential recipients. Furthermore, Enterococcus faecalis inhibitory peptides are thought to be competitive inhibitors of specific mating pheromones (50). There is no evidence that a specific peptide stimulates transfer of ICEBs1 or competes with the inhibitory PhrI peptide for binding to RapI. Hence, multiple molecular mechanisms evolved to inhibit self-transfer of mobile genetic elements using secreted signaling molecules.

Many lysogenic bacteriophages (51) and the ICE SXT (14) are induced by the SOS response. We suspect that the SOS response inactivates the immunity repressor of ICEBs1, because that is how the SOS response induces some other mobile genetic elements (14, 51). However, further work will be needed to reveal the molecular mechanisms regulating SOS-mediated induction of ICEBs1.

Rap-Phr Systems in Other Bacillus Mobile Elements. In addition to the chromosomally encoded rap-phr cassettes in Bacillus species, rap-phr cassettes are found on the B. subtilis plasmids pTA1060, pTA1040, pPOD2000, and pLS20, the B. licheniformis plasmids pFL5 and pFL7, the B. cereus plasmid pBC10987, the B. subtilis phage φ105, the defective B. subtilis prophage skin, and the B. anthracis bacteriophage λBa04 (see Table 6, which is published as supporting information on the PNAS web site). rap60 and phr60, from pTA1060, have been characterized. Rap60 inhibits degradative enzyme production; this inhibition is antagonized by Phr60 (52). Rap60 and Phr60 were studied in the absence of pTA1060, and their effects on mobility of pTA1060 were not reported. To our knowledge, the remaining rap-phr systems contained on mobile elements (other than rapE and rapI) have not been characterized. We postulate that these raps and phrs might regulate the mobility of their respective genetic elements, thereby modulating horizontal gene transfer and bacterial evolution.