Regulation of a Bacillus Subtilis Mobile Genetic Element by Intercellular Signaling and the Global DNA Damage Response

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

Jennifer M. Auchtung, Catherine A. Lee, Rita E. Monson*, Alisa P. Lehman, and Alan D. Grossman†

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, July 12, 2005 (received for review June 16, 2005) Horizontal gene transfer contributes to the evolution of bacterial Phr peptides directly inhibit the activities of intracellular regu- species. Mobile genetic elements play an important role in hori- lators, known as Rap proteins (20–24) (Fig. 1). The character- zontal gene transfer, and characterization of the regulation of ized Rap proteins directly (24, 25) or indirectly (23, 26) inhibit these elements should provide insight into conditions that inﬂu- the activities of transcription factors that regulate sporulation, ence bacterial evolution. We characterized a mobile genetic ele- competence development, and production of degradative en- ment, ICEBs1, in the Gram-positive bacterium Bacillus subtilis and zymes and antibiotics (20, 22–24). found that it is a functional integrative and conjugative element RapI and PhrI are encoded by ICEBs1. We found that RapI (ICE) capable of transferring to Bacillus and Listeria species. We activates ICEBs1 gene expression, excision, and transfer and that identiﬁed two conditions that promote ICEBs1 transfer: conditions the PhrI peptide antagonizes the activity of RapI. Furthermore, that induce the global DNA damage response and crowding by expression of rapI and phrI is stimulated by conditions of low potential recipients that lack ICEBs1. Transfer of ICEBs1 into cells nutrient availability and high cell density. This combined regu- that already contain the element is inhibited by an intercellular lation activates ICEBs1 excision and transfer when host cells are signaling peptide encoded by ICEBs1. The dual regulation of ICEBs1 crowded by potential recipients that lack ICEBs1 and do not allows for passive propagation in the host cell until either the produce the PhrI peptide. potential mating partners lacking ICEBs1 are present or the host In addition, we observed that the global DNA damage (SOS) cell is in distress. response activates ICEBs1 excision and transfer, independently of rapI and phrI. Therefore, at least two conditions promote conjugation ͉ horizontal gene transfer ͉ quorum sensing ͉ peptide ICEBs1 excision and transfer: the presence of a high concen- signaling ͉ DNA microarrays tration of cells lacking ICEBs1 and host cell distress. In the absence of these conditions, ICEBs1 is propagated by the host orizontal gene transfer and mobile genetic elements play a through vertical gene transfer to progeny cells. Hsignificant role in bacterial evolution (1–4). Conjugative transposons (5, 6), also known as integrative and conjugative Materials and Methods elements (ICEs) (4, 7), are mobile genetic elements that are Media. Cells were grown at 37°C, with agitation in LB medium normally integrated into the chromosome. They can excise and (27), defined minimal medium (28) (supplemented with re- transfer to recipients through conjugation (mating) and inte- quired amino acids when necessary), Schaeffer’s nutrient broth grate into the chromosome of the recipient (5, 6). ICEs encode sporulation medium (29), or brain heart infusion medium (29), proteins required for conjugal transfer and can also encode as indicated. Antibiotics and other chemicals were used at the proteins involved in resistance to antibiotics (5, 6), metabolism following concentrations: ampicillin (100 ␮g͞ml), chloramphen- of alternative carbon sources (4, 8), symbiosis (9), and other icol (5 ␮g͞ml), kanamycin (5 ␮g͞ml), spectinomycin (100 ␮g͞ processes (10). ICEs and putative ICEs have been found in many ml), streptomycin (100 ␮g͞ml), erythromycin (0.5 ␮g͞ml), and bacteria (10) and are important agents of horizontal gene lincomycin (12.5 ␮g͞ml) together, to select for macrolide- transfer because they are capable of moving themselves and lincosamide-streptogramin B resistance, and isopropyl-␤-D- other DNA to recipients (6, 11–13). thiogalactopyranoside (IPTG) (1 mM, Sigma) and mitomycin C Mechanisms that regulate transfer have been determined for (MMC) (1 ␮g͞ml, Sigma). several ICEs. In some cases, an antibiotic induces transfer of an element that encodes resistance to that antibiotic (5, 6, 14). Strains and Alleles. Strains used in this study are listed in Table 3, Transfer of the Streptomyces ICE pSAM2 is inhibited by the which is published as supporting information on the PNAS web presence of a pSAM2-encoded protein in the recipient (15). site. The Escherichia coli strain used for cloning is an MC1061 Recently, it was shown that the DNA damage response stimu- derivative carrying FЈ(lacIq) lacZM15 Tn10 (tet). Standard tech- lates transfer of SXT, an ICE from Vibrio cholerae (14). niques were used for cloning and strain construction (27, 29). We characterized a 20-kb ICE, ICEBs1 (16), in Bacillus subtilis For overexpression in B. subtilis, rapI, phrI, and rapI phrI and found that ICEBs1 excision and transfer is regulated by a were cloned downstream of the IPTG-inducible promoters secreted peptide encoded by ICEBs1. Pspank(hy) (30) or Pspank (28), both generous gifts from D. Many Gram-positive bacteria use secreted signaling peptides Rudner (Harvard Medical School, Boston), and integrated 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 Abbreviations: att, attachment; ICE, integrative and conjugative element; IPTG, isopropyl- ␤-D-thiogalactopyranoside; MMC, mitomycin C; Opp, oligopeptide permease; SOS, global encoded by phr genes (reviewed in ref. 18). It has been suggested DNA damage. that Phr peptides act as autocrine signals and not in cell–cell *Present address: Department of Biochemistry, University of Cambridge, Cambridge signaling (reviewed in ref. 19), although this is clearly not true CB2 1QW, United Kingdom. for all Phr peptides (20, 21). Nonetheless, all characterized Phr †To whom correspondence should be addressed at: Department of Biology, Building peptides have a common mechanism of action. After secretion 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139. E-mail: and extracellular accumulation, Phr pentapeptides are imported [email protected]. through the oligopeptide permease (Opp); once inside the cell, © 2005 by The National Academy of Sciences of the USA

12554–12559 ͉ PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505835102 Downloaded by guest on September 25, 2021 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 3Ј end of a leucyl-tRNA gene (trnS-leu2). The black arrow indicates int, Fig. 1. Phr peptide signaling in B. subtilis. rap and phr genes are transcribed encoding the putative integrase. The hatched arrow indicates immR, encod- and translated (A); pre-Phr peptides are secreted and processed (B); mature ing the putative immunity repressor. Shaded arrows indicate genes similar to Phr peptides are transported into the cell by the Opp (C); once inside the cell, those found in other ICEs (16). The numbers below the cartoon of ICEBs1 Phr peptides inhibit the activities of regulators known as Rap proteins (D); indicate the mean fold-increase in mRNA levels in cells overexpressing rapI. each characterized Rap protein inhibits the activity of a transcription factor, Pspank(hy)-rapI (JMA28) cells were grown for at least four generations to either directly or indirectly (E); and inhibition of transcription factors lead to midexponential phase in minimal medium. IPTG was added to half of the cellular responses (F). 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 into the amyE locus by homologous recombination. Pspank identified, as described in Materials and Methods. Expression of the three and Pspank(hy) (with no inserts) were also integrated into genes at the left end did not change significantly nor did the expression of amyE. almost all chromosomal genes. Experimental details and additional microarray results are in Table 4 and Supporting Text, which are published as sup- The rapI–lacZ promoter fusion was generated by cloning the porting information on the PNAS web site. 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. treatment). PCR with the primer pair oJMA93 and oJMA100 Isolation of spontaneous streptomycin-resistant mutants and detected the chromosomal junction formed after ICEBs1 exci- construction of the following alleles is described in Supporting sion. PCR with the primer pair oJMA95 and oJMA97 detected Methods, which is published as supporting information on the the excised ICEBs1 circle. Primer sequences, PCR conditions, PNAS web site: ICEBs1::kan, an insertion of a kanamycin- and cycling parameters are described in Supporting Methods. Ј resistance gene between the 3 end of yddM and attachment (att) Products were visualized on 2% agarose gels stained with site attR; ICEBs10, a complete loss of ICEBs1 that leaves the ⌬ ethidium bromide. PCR was performed on at least two inde- chromosomal att site intact; and (ICEBs1)206::cat, a deletion pendent biological replicates. Representative results are shown. of the entire ICE, including attR, and insertion of a chloram- For linear-range (quantitative) PCR, known concentrations of phenicol-resistance gene. Null mutations included ⌬(rapI ⌬ ⌬ DNA were diluted serially, and regions were amplified by using phrI)342::kan, int205::cat, and immR208::cat. S. Branda and the indicated primer pairs. Products were visualized on 2% ⌬phrI173 erm ⌬ rapI R. Kolter generously provided :: and ( agarose gels stained with ethidium bromide and quantified by phrI)260::erm. using the ChemiImager gel documentation system (Alpha In- comK::spc and comK::cat (32), ⌬abrB::cat (33), recA260 (34, notech, San Leandro, CA). Reactions were deemed in the linear 35), and opp::cat [opp::Tn917lac::pTV21⌬2cat (opp ϭ spo0K)] range when three 2-fold serial dilutions of input DNA produced (36), were described previously. linearly decreasing amounts of PCR product.

The relative increase in excision is reported for circular MICROBIOLOGY DNA Microarrays. Cells were harvested, and total RNA was prepared as described in ref. 30. RNA from each sample was intermediate PCR products. Fold-increase was determined by reverse-transcribed and labeled with Cy3 or Cy5. Labeled sam- calculating the amount of PCR product of the ICEBs1 circle in ples were combined and purified with Qiagen PCR purification each experimental sample, compared with the amount of columns and hybridized to microarrays containing PCR products ICEBs1 circle PCR product from the control sample for each of virtually all of the B. subtilis ORFs (30). Similar hybridization experiment. These fold-increases were normalized to the amount experiments were performed by using microarrays containing a of PCR product from cotF for each sample. cotF, a chromosomal unique DNA oligonucleotide for each B. subtilis ORF. Addi- site unaffected by ICEBs1 excision, was amplified with primers tional details are described in Supporting Methods. oLIN93 and oLIN94 (38). The fold-increase is reported as the Ϯ Arrays were scanned and analyzed with the program GENEPIX mean ( SEM) from at least two independent experiments. 3.0 (Axon Instruments, Union City, CA). Cy3 and Cy5 signals for In experiments with mixed cultures, an additional normalization each spot were normalized to the total Cy3 and Cy5 signals of the was done to take into account only the cells capable of excision of array and were obtained for each spot that had a signal above ICEBs1. PCR was also done with the primer pair oJMA177 and background for 50% of pixels. Iterative outlier analysis (30, 37) oJMA178 that amplifies DNA [amyE::Pspank(hy)] unique to the was used to identify spots (genes) whose experimental mean population of cells capable of excision. The amount of this product ratio was Ͼ2.5 SDs away from the mean ratio of the population in the experimental sample was compared with the amount of this of genes in the third iteration of the calculation (outlier cutoff). product in the control to determine the number of cells in the The probability that the mean ratios of these outliers were experimental sample capable of excision. All cells in the control greater than the outlier cutoff was calculated by using the normal were capable of excision and contained amyE::Pspank(hy). distribution function for each spot; those genes with Ն95% probability were considered significantly changed. The mean Mating Experiments. Donors and recipients were grown in LB (for ratio for a set of triplicate experiments is reported. matings with Bacillus) or brain heart infusion (BHI) medium (for matings with Listeria) when assaying transfer from cells overex- Excision Assays. DNA was extracted by using the Qiagen DNEasy pressing rapI or in defined minimal medium when assaying tissue kit (protocol for Gram-positive bacteria with RNase A transfer from cells treated with MMC. ICEBs1 excision in donor

Auchtung et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12555 Downloaded by guest on September 25, 2021 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 ⌬(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 ␮M. 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 ␮M 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 ⌬(rapI phrI), JMA168], defined as 100%; black bar, overexpression of rapI and phrI [Pspank(hy)-(rapI phrI) ⌬(rapI phrI), JMA186]; shaded bar, overexpression of rapI in an opp-null mutant [Pspank(hy)-rapI ⌬(rapI phrI) ⌬opp, CAL51]; hatched bar, overexpression of rapI and phrI in an opp-null mutant [Pspank(hy)-(rapI phrI) ⌬(rapI phrI) ⌬opp, CAL52]. (E) Excision of ICEBs1 increases in a phrI-null mutant. Cells were grown in nutrient broth sporulation medium. Samples were collected from cells Ϸ2 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, ⌬phrI (SSB173); shaded bar, ⌬(rapI phrI) (SSB260); hatched bar, ⌬phrI Pspank(hy)-phrI (JMA298). Ϫ͞c indicates complementation of ⌬phrI mutation.

cells was induced either by overexpression of rapI [Pspank(hy)- comparative sequence analysis (16). ICEBs1 is flanked by 60-bp rapI ⌬(rapI phrI), strain JMA168] or addition of MMC [⌬(rapI direct repeats, the likely att sites. One of the potential att sites is phrI), strain IRN342]. At1hafterinduction, equal volumes of in the 3Ј end of a tRNA gene, a common integration site for donor and recipient cultures were mixed and filtered onto a mobile elements (41). ICEBs1 contains int (previously ydcL) sterile nitrocellulose filter (0.2 ␮m pore size, Nalgene), placed on (16), encoding a putative ␭-like integrase, immR (previously LB or BHI agar plates, and incubated at 37°C for Ϸ3 h. Cells ydcN), encoding a putative bacteriophage-like immunity repres- were removed from filters by washing with 5 ml of Spizizen sor with 50% amino acid similarity to the repressor of B. subtilis minimal salts (29). Transconjugants were isolated by selecting phage ␾105 (42, 43), and seven genes similar to genes from other for antibiotic resistance unique to the recipient and the kana- ICEs (16). Our results demonstrate that RapI activates ICEBs1 mycin resistance in ICEBs1. Donor and recipient numbers were gene expression. This activation is most likely by directly or also determined by selective plating. Concentrated, unmixed indirectly inhibiting the activity of the putative immunity repres- donor and recipient cultures spread on the double-antibiotic sor ImmR (J.M.A. and A.D.G., unpublished data). Furthermore, agar did not give rise to spontaneous antibiotic-resistant mu- activation of ICEBs1 gene expression is specific to overexpres- tants. Transfer of DNA to the donor through transformation was sion of rapI, because overproduction of other B. subtilis Rap not observed. proteins did not stimulate ICEBs1 gene expression (J.M.A. and Mating frequencies were calculated by dividing the number of A.D.G., unpublished results). transconjugants by the number of donor cells, except in the case of donor cells treated with MMC, where mating frequencies were ICEBs1 Excises and Transfers. Before conjugal transfer, an ICE calculated relative to recipients. The reported transfer frequen- excises from the chromosome, forming a circular intermediate cies are the mean (ϮSEM) of at least two independent biological and a repaired chromosomal junction (5). We used a PCR-based replicates. assay to detect products formed upon ICEBs1 excision (Fig. 3A). We detected a low level of circular ICEBs1 intermediates and ␤-Galactosidase Assays. ␤-Galactosidase-specific activity of a rap- repaired chromosomal junctions in control cells of B. subtilis I–lacZ fusion was assayed throughout growth of wild-type and (Fig. 3B), indicating that excision occurs at a low level in this ⌬abrB cultures in sporulation media, as described in ref. 39. population of cells. Overexpression of rapI greatly stimulated ICEBs1 excision (Fig. 3B). Because expression of the putative Results and Discussion integrase is not activated by rapI overexpression, RapI likely Identification of a Mobile Genetic Element Regulated by Peptide stimulates excision by activating expression of an accessory Signaling. B. subtilis encodes seven phr genes (40), each located protein required for excision. Many integrase proteins require in an operon with a rap gene. To identify biological processes accessory proteins for excision (44). regulated by the uncharacterized rapI-phrI operon, we used rapI overexpression also stimulated ICEBs1 transfer to recip- whole-genome DNA microarrays to monitor changes in mRNA ients. To assay transfer from donor cells, we replaced rapI and levels caused by overexpression of rapI from an IPTG-inducible phrI with an antibiotic-resistance marker. Deletion of rapI and promoter [Pspank(hy)-rapI]. phrI had minimal effects on the excision of ICEBs1 in wild-type In two types of microarray experiments, overproduction of cells (Fig. 3E) and in cells overexpressing rapI (data not shown). RapI caused mRNA levels of 18 genes to increase Ն4-fold (Fig. We assayed transfer of ICEBs1 on a solid surface (filter mating) 2; and see Table 4). All 18 genes cluster around rapI and phrI and by mixing donor cells [Pspank(hy)-rapI ⌬(rapI phrI)::kan], in are in the 20-kb ICEBs1 element (Fig. 2) previously identified by which rapI overexpression had been induced for 1 h, with an

12556 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505835102 Auchtung et al. Downloaded by guest on September 25, 2021 Table 1. Frequency of ICEBs1 mating into recipients fold, relative to wild-type cells (Fig. 3E). This increase required ⌬ Recipient Mating frequency* RapI; excision in (rapI phrI) cells was similar to wild-type (Fig. 3E). Ectopic expression of phrI complemented the ⌬phrI phe- B. subtilis ICEBs10 (CAL89) 1 ϫ 10Ϫ2 Ϯ 3 ϫ 10Ϫ3 notype, reducing ICEBs1 excision back to a low level (Fig. 3E), B. subtilis ICEBs1ϩ (CAL88) 2 ϫ 10Ϫ4 Ϯ 1 ϫ 10Ϫ4 indicating that increased excision in the ⌬phrI mutant was due to B. anthracis (UM44-1C9) 6 ϫ 10Ϫ3 Ϯ 5 ϫ 10Ϫ3 loss of phrI and not to effects on neighboring genes. B. licheniformis (REM42) 2 ϫ 10Ϫ4 Ϯ 5 ϫ 10Ϫ6 L. monocytogenes (10403S) 8 ϫ 10Ϫ6 Ϯ 6 ϫ 10Ϫ6 Regulation of ICEBs1 Excision and Transfer by Intercellular Signaling. *Mating was assayed 1 h after induction of rapI overexpression from donor The preceding results indicated that PhrI peptide signaling cells (Pspank(hy)-rapI ⌬(rapI phrI)ϻkan, JMA168). Mating frequency is the inhibited ICEBs1 excision but did not indicate whether the PhrI number of transconjugants per donor (ϮSEM). 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 equal number of recipient B. subtilis cells that lacked ICEBs1 inhibited when the concentration of PhrI peptide produced by (ICEBs10). the population of cells is high, as when the majority of cells in the ICEBs1 transferred at an average frequency of Ϸ1 ϫ 10Ϫ2 population contain ICEBs1 and produce PhrI. However, when transconjugants (recipients that received ICEBs1) per donor the concentration of PhrI peptide is low, as when the majority of (Table 1). Transfer into recipients that contained ICEBs1 oc- cells in the population lack ICEBs1 and do not produce the PhrI curred with Ϸ50-fold lower frequency (Table 1), indicating that peptide, then RapI-dependent activation of ICEBs1, excision, ICEBs1 encodes at least one mechanism that inhibits acquisition and transfer should occur. ICEBs1 could use this mechanism to of a second element. Acquisition of ICEBs1 by recipients was not inhibit excision and transfer when surrounded by cells that due to natural transformation, because the recipients were comK already contain ICEBs1. mutants incapable of transformation (32). Transfer of ICEBs1 To test this model, we monitored excision in a minority from nonactivated donor cells [⌬(rapI phrI)::kan, IRN342] was population of ICEBs1ϩ cells when they were grown together with not detected under these conditions (Ͻ2 ϫ 10Ϫ8 transconjugants a majority of ICEBs1-containing cells that either produced PhrI per donor). (phrIϩ) or did not produce PhrI (⌬phrI) (Fig. 4A). In these mixed cultures, only the minority ICEBs1ϩ cells were capable of Transfer of ICEBs1 into Bacillus and Listeria Recipients. The putative excision, because cells in the majority lacked integrase (⌬int), bacterial chromosomal att site of ICEBs1 is conserved (Ն52 of which is required for ICEBs1 excision (C.A.L. and A.D.G., 60 base pairs identical) in Bacillus, Listeria, and Staphylococcus unpublished results). species (see Fig. 5, which is published as supporting information During midexponential growth, ICEBs1 excision was low, on the PNAS web site). We assayed transfer of ICEBs1 from whether minority ICEBs1ϩ cells were grown with excess phrIϩ or B. subtilis donor cells overexpressing rapI into Bacillus anthracis, ⌬phrI cells (Fig. 4B). However, Ϸ2 h after the cells entered Bacillus licheniformis, and Listeria monocytogenes and found that stationary phase, ICEBs1 excision was stimulated Ͼ40-fold in ICEBs1 mated into all three species (Table 1). The efficient the ICEBs1ϩ cells mixed with ⌬phrI cells, relative to ICEBs1ϩ transfer of ICEBs1 into Bacillus and Listeria species, and, cells mixed with phrIϩ cells (Fig. 4B). We observed a similar potentially, Staphylococcus species (not tested), indicates that increase in excision when ICEBs1ϩ cells were mixed with cells ICEBs1 may be a useful tool to facilitate genetic manipulation lacking ICEBs1 (data not shown). of these organisms. These results indicate that the PhrI peptide acts as an intercellular signaling peptide that inhibits ICEBs1 excision when Inhibition of ICEBs1 Excision by the PhrI Peptide. Because the cells are crowded by cells that contain ICEBs1 and produce the activities of the characterized Rap proteins are inhibited by their PhrI peptide. Furthermore, ICEBs1 excision is inhibited in cognate Phr peptides and rapI overexpression activates ICEBs1 exponential growth, irrespective of whether cells in the majority excision and transfer, we investigated whether PhrI peptide population contain phrI, indicating that an additional mecha- MICROBIOLOGY signaling inhibits ICEBs1 excision and transfer. Excision of nism inhibits ICEBs1 excision and transfer. AbrB is a transition- ICEBs1 in cells overexpressing rapI was inhibited by the addition state regulator that represses transcription of several B. subtilis of synthetic PhrI peptide (Fig. 3C). The active PhrI peptide, the genes during exponential phase and is inactive under conditions five C-terminal amino acids of the 38-aa precursor protein, was of nutrient limitation and high cell density (reviewed in ref. 46). predicted based on its similarity to characterized Phr peptides We found that transcription of rapI, measured with a rapI–lacZ (18, 19). The addition of 1 ␮M synthetic PhrI peptide inhibited promoter fusion, increased Ϸ5-fold in an abrB mutant (CAL26), RapI-dependent excision of ICEBs1 Ϸ20-fold, and addition of relative to wild-type cells (CAL15), indicating that AbrB re- 100 nM PhrI peptide inhibited excision Ϸ3-fold (Fig. 3C). These presses rapI transcription, either directly or indirectly. Consistent concentrations of peptide are similar to the biologically active with this model, we also found that ICEBs1 excision increased in concentrations of other Phr peptides (21, 23, 45). These results ⌬abrB cells, relative to wild-type cells; this effect was much larger demonstrate that the PhrI pentapeptide inhibits RapI- in exponential phase than in stationary phase (see Fig. 6, which dependent activation of ICEBs1 excision. is published as supporting information on the PNAS web site). Excision of ICEBs1 in cells overexpressing rapI was inhibited Taken together, these observations indicate that at least two Ϸ50-fold by cooverexpression of phrI (Fig. 3D). This inhibition mechanisms regulate RapI-dependent activation of ICEBs1 depended on the presence of the opp, a transporter required for excision. When nutrients are abundant and cell density is low, uptake of Phr peptides (20, 22, 23). Excision occurred at similar AbrB represses rapI transcription, preventing RapI-dependent levels in oppϪ cells cooverexpressing rapI and phrI and in oppϩ activation of ICEBs1 excision. As cells enter stationary phase, cells overexpressing rapI alone (Fig. 3D). These data provide rapI transcription is derepressed and RapI can activate excision further evidence that the secreted PhrI peptide is imported but only when the concentration of PhrI peptide is too low to through the Opp and inhibits RapI-dependent activation of inhibit RapI. ICEBs1 excision. As expected, transfer of ICEBs1 was also inhibited when PhrI also inhibits ICEBs1 excision when rapI is expressed from potential donors were surrounded by cells that produced the its native promoter. Deletion of the gene encoding PhrI (⌬phrI), PhrI peptide. We introduced an antibiotic-resistance cassette in otherwise wild-type cells, activated ICEBs1 excision Ͼ5,000- into ICEBs1 between the last gene of the element (yddM) and the

Auchtung et al. PNAS ͉ August 30, 2005 ͉ vol. 102 ͉ no. 35 ͉ 12557 Downloaded by guest on September 25, 2021 Table 2. Transfer of ICEBs1 is inhibited if the surrounding cells are phrI؉ Recipient

ICEBs1ϩ ICEBs1ϩ Donor ExcisionϪ PhrIϩ ExcisionϪ PhrIϪ

rapIϩ phrIϩ 1.0 ϫ 10Ϫ5 Ϯ 4.0 ϫ 10Ϫ6 3.0 ϫ 10Ϫ3 Ϯ 1.0 ϫ 10Ϫ3 ⌬(rapIphrI) 1.0 ϫ 10Ϫ5 Ϯ 4.0 ϫ 10Ϫ6 5.0 ϫ 10Ϫ6 Ϯ 2.0 ϫ 10Ϫ6

A minority population of ICEBs1-containing cells (potential donors), containing an antibiotic-resistance gene in ICEBs1(ICEBs1ϻkan rapIϩ phrIϩ, 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 phrIϩ (phrIϩ ⌬int comK, JMA381) or phrIϪ (⌬phrI ⌬int comK, JMA306), as described in Fig. 4. To show depen- dence on rapI in the donor, a similar experiment was done with potential donors lacking rapI and phrI[⌬(rapI phrI)ϻkan, 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 Ϸ1 potential donor to 24 potential recipients (total OD600 Ϸ0.015–0.03) and were Fig. 4. Excision is inhibited in the presence of PhrIϩ cells. (A) Outline of grown in coculture until Ϸ2 h after entry into stationary phase. A 5-ml aliquot mixing experiments. A minority population (Ϸ4% of total) of cells capable of of each coculture was removed, mixed with 7.5 ml of fresh medium, filtered, ICEBs1 excision and transfer (Excisionϩ PhrIϩ) was mixed with a majority and incubated on sporulation medium agar for Ϸ3 h. Filters were washed, and population (Ϸ96% of total) of cells incapable of ICEBs1 excision and transfer samples were plated selectively, as described in Materials and Methods. The that either did (ExcisionϪ PhrIϩ) or did not (ExcisionϪ PhrIϪ) encode PhrI. (B) mean number of transconjugants per donor cell (ϮSEM) for at least two Excision of ICEBs1 in cells grown in mixed culture with a majority of ICEBs1 independent experiments is reported. ICEBs1 transfer occurred much more ExcisionϪ PhrIϩ (JMA205, open bars) or ICEBs1 ExcisionϪ PhrIϪ (JMA304, black efficiently under these mating conditions than under the conditions described bars) cells was measured during exponential growth and Ϸ2 h after the entry in Table 1 (see Table 5, which is published as supporting information on the into stationary phase. Cells were grown separately in nutrient broth sporula- PNAS web site, and Supporting Text. tion medium to midexponential phase. Cells were diluted into fresh medium at a ratio of Ϸ1 minority cell [JMA35, Pspank(hy)] to 24 majority cells [JMA205 ⌬ ⌬ ⌬ Ϸ ( int) or JMA304 ( int phrI)] to a total OD600 of 0.015–0.03 and were conditions that induce the SOS response (A. Goranov, E. cocultured throughout growth. Samples were collected during midexponen- Kuester-Schoeck, R. Britton, and A.D.G., unpublished results). tial growth (OD600 Ϸ0.2) and Ϸ2 h after cells entered stationary phase and were used for linear-range PCR assays. In addition to the circular intermediate Treatment of wild-type cells with MMC, a DNA-damaging agent and chromosomal control (cotF) primer pairs (see Materials and Methods), the that induces the SOS response in B. subtilis (47), stimulated primer pair oJMA177 and oJMA178 was used in linear-range PCR assays to ICEBs1 excision (Fig. 3B). Increased gene expression and exci- amplify a sequence specific to Pspank(hy), which is present in only the minority sion in response to MMC depended on recA, which is required JMA35 cells. The amount of circular intermediate product from each experi- for the SOS response (47), and was independent of rapI and phrI mental sample was normalized to the amount of Pspank(hy) and cotF products (data not shown). in that sample. This ratio was normalized to the amount of circular interme- Mating frequency also increased when potential donor cells diate 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, [ (rapI phrI)::kan, IRN342] were treated with MMC. The mean ϫ Ϫ4Ϯ ϫ Ϫ5 data not shown) to give the relative increase in excision. mating frequency was 2 10 8 10 transconjugants per ICEBs10 recipient (CAL89). Mating was undetectable from untreated cells under these conditions (Ͻ2 ϫ 10Ϫ8 transconju- attachment site attR. This insertion did not have a significant gants per recipient). Mating frequency was determined relative effect on mating frequency; donor cells overexpressing rapI that to recipients, because MMC treatment reduced the viability of contained this insertion (JMA448) or an antibiotic insertion in donors. Induction of ICEBs1 excision and transfer by the SOS rapI and phrI (JMA168) mated at similar frequencies (data not response may be an attempt by the element to escape the shown). We tested transfer of ICEBs1 from a minority popula- distressed cell for a viable host. tion (ICEBs1::kan) into cells in the majority population that either did (phrIϩ ⌬int comK) or did not (⌬phrI ⌬int comK) Conserved Signals Regulate Dissemination of Mobile Genetic Ele- produce PhrI. ICEBs1 transfer in the mixed cultures, measured ments. We determined that ICEBs1 gene expression, excision, 2 h after cells entered stationary phase, was Ͼ100-fold higher and transfer are inhibited by a self-encoded peptide and acti- into recipients that lacked phrI than into cells that contained phrI vated by the SOS response. Intercellular signaling also regulates (Table 2). This stimulation depended on RapI; it did not occur transfer of some conjugative plasmids. Two well studied exam- when the donor cells lacked rapI and phrI (Table 2). ples are transfer of the Ti plasmid in Agrobacterium tumafaciens Taken together, the results of the excision and mating exper- (reviewed in ref. 48) and transfer of pheromone-inducible iments indicate that ICEBs1 excision and transfer is more active plasmids in Enterococcus faecalis (reviewed in refs. 49 and 50). when cells are crowded by potential mating partners that do not Ti plasmid transfer is stimulated by the presence of cells that produce the PhrI peptide. Excision and transfer is limited to contain the plasmid; this stimulation depends on the plasmid- conditions that are likely to correlate with cell crowding, star- encoded signal synthetase TraI, which synthesizes 3-oxo-8 ho- vation, and high cell density, through the growth-phase- moserine lactone, and the plasmid-encoded regulatory protein dependent regulation of rapI transcription. In this way, ICEBs1 TraR (48). In contrast, transfer of ICEBs1 is inhibited by the uses intercellular peptide signaling to coordinate excision and presence of cells that contain the element. mating with conditions that favor its productive dissemination to In Enterococcus faecalis, several mating pheromones (pep- recipients lacking ICEBs1. tides) are encoded in the chromosome. Each pheromone stimulates transfer of a specific conjugal plasmid, and production of Activation of ICEBs1 Excision and Transfer by the SOS Response. these pheromones by cells lacking specific plasmids stimulates Previous analysis of mRNA levels using DNA microarrays transfer of those plasmids from donors (49, 50). Plasmid- indicated that genes in ICEBs1 are activated by a variety of containing cells also produce unique plasmid-encoded peptides

12558 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505835102 Auchtung et al. Downloaded by guest on September 25, 2021 that inhibit plasmid transfer to potential recipients that already cies, rap–phr cassettes are found on the B. subtilis plasmids contain the plasmid (49, 50). pTA1060, pTA1040, pPOD2000, and pLS20, the B. lichenifor- Although peptides produced by Enterococcus faecalis phero- mis plasmids pFL5 and pFL7, the B. cereus plasmid pBC10987, mone-responsive plasmids and ICEBs1 both inhibit transfer, the the B. subtilis phage ␾105, the defective B. subtilis prophage regulatory mechanisms are different. With Enterococcus faecalis skin, and the B. anthracis bacteriophage ␭Ba04 (see Table 6, plasmids, specific peptide signals produced by recipients trigger which is published as supporting information on the PNAS web transfer from donor cells. ICEBs1 transfer is stimulated by site). rap60 and phr60, from pTA1060, have been character- conditions (low nutrient availability and high cell density) likely ized. Rap60 inhibits degradative enzyme production; this to correlate with a high number of potential recipients. Further- inhibition is antagonized by Phr60 (52). Rap60 and Phr60 were more, Enterococcus faecalis inhibitory peptides are thought to be studied in the absence of pTA1060, and their effects on competitive inhibitors of specific mating pheromones (50). mobility of pTA1060 were not reported. To our knowledge, the There is no evidence that a specific peptide stimulates transfer remaining rap–phr systems contained on mobile elements (other than rapE and rapI) have not been characterized. We of ICEBs1 or competes with the inhibitory PhrI peptide for postulate that these raps and phrs might regulate the mobility binding to RapI. Hence, multiple molecular mechanisms evolved of their respective genetic elements, thereby modulating hor- to inhibit self-transfer of mobile genetic elements using secreted izontal gene transfer and bacterial evolution. signaling molecules. Many lysogenic bacteriophages (51) and the ICE SXT (14) are We thank S. Branda, M. Bucknor, and R. Kolter (Harvard Medical induced by the SOS response. We suspect that the SOS response School, Boston) for providing strains and sharing data before publica- inactivates the immunity repressor of ICEBs1, because that is tion; D. Rudner (Harvard Medical School) for Pspank and Pspank(hy); how the SOS response induces some other mobile genetic D. Higgins (Harvard Medical School), T. Koehler (University of Texas elements (14, 51). However, further work will be needed to Medical School, Houston), and E. Ryan (Massachusetts General Hos- reveal the molecular mechanisms regulating SOS-mediated in- pital, Boston) for providing strains and advice for working with L. monocytogenes and B. anthracis; and A. Amon, M. Berkmen, M. Rokop, R. duction of ICEBs1. Sauer, and F. Solomon for comments on the manuscript. This work was supported, in part, by National Institutes of Health (NIH) Public Health Rap–Phr Systems in Other Bacillus Mobile Elements. In addition to Service Grant GM50895 (to A.D.G.). J.M.A. was supported, in part, by the chromosomally encoded rap–phr cassettes in Bacillus spe- a NIH predoctoral training grant.

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