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. 2005 Aug 30;102(35):12554-9.
doi: 10.1073/pnas.0505835102. Epub 2005 Aug 16.

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

Affiliations

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

Jennifer M Auchtung et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
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).
Fig. 2.
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.
Fig. 3.
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.
Fig. 4.
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.

References

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