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

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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 microar- ray 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
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