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

Auchtung et al. 10.1073/pnas.0505835102.

Supporting Information

Files in this Data Supplement:

Supporting Table 3
Supporting Text
Supporting Table 4
Supporting Figure 5
Supporting Figure 6
Supporting Table 5
Supporting Table 6




Supporting Figure 5

Fig. 5. ICEBs1 att site is found in other Gram-positive species. Sequences closely related to the 60-bp direct repeat sequence were identified through BLAST (1) and were aligned with the B. subtilis sequence. Consensus nucleotides are identified by a dash. Nucleotides that diverge from the B. subtilis sequence, including missing nucleotides, are underlined and in boldface type.

1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–3402.

Supporting Figure 6

Fig. 6. AbrB inhibits ICEBs1 excision. Wild-type (JH642, black bar) and ΔabrB (AG839, white bar) cells were grown in DSM. Samples were collected from cells during exponential phase (OD600 ≈0.2) and ≈2 h after the entry into stationary phase. Excision of ICEBs1 was determined by linear-range PCR and was normalized to the amount of excision in wild-type cells during exponential phase.

Table 3. Strains used in this study

*All strains through JMA448 are derived from JH642 and contain the trpC and pheA mutations.

1. Perego, M., Spiegelman, G. B., & Hoch, J. A. (1988) Mol. Microbiol. 2, 689–699.

2. Lemon, K. P., Kurtser, I., & Grossman, A. D. (2001) Proc. Natl. Acad. Sci. USA 98, 212–217.

3. Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., & Kolter, R. (2001) Proc. Natl. Acad. Sci. USA 98, 11621–11626.

4. Hoffmaster, A. R. & Koehler, T. M. (1997) Infect. Immun. 65, 3091–3099.

5. Bishop, D. K. & Hinrichs, D. J. (1987) J. Immunol. 139, 2005–2009.

Table 4. Changes in mRNA levels caused by overexpression of rapI

*Description of protein functions are derived from http://genolist.pasteur.fr/SubtiList. Homology with Tn916 genes was determined through BLAST analysis (4).

^†The average fold-change in gene expression in the rapI-overexpressing cells, relative to control cells from each set of triplicate experiments is shown. Positive values indicate increased expression in rapI-overexpressing cells, and negative values indicate decreased expression.

^‡Genes regulated by Spo0A (directly or indirectly) (1–3). Repression of genes in the Spo0A regulon is consistent with an observed reduction in sporulation caused by rapI overexpression (6).

^§Genes that are part of ICEBs1.

^¶ydcQ was identified as encoding a putative DNA translocase because of the presence of a conserved FtsK/SpoIIIE-like domain identified by the program SMART (5).

^||rapIrapI

**We do not report the fold-change in mRNA levels for phrI because the arrays do not distinguish between the endogenous phrI and the partial fragment of the phrI transcript that is overexpressed from the ectopic rapI construct. The 5′ end of phrI overlaps the 3′ end of rapI by 41 nucleotides.

1. Fawcett, P., Eichenberger, P., Losick, R. & Youngman, P. (2000) Proc. Natl. Acad. Sci. USA 97, 8063–8068.

2. Piggot, P. J. & Losick, R. (2002) in Bacillus Subtilis and its Closest Relatives from Genes to Cells, eds. Sonenshein, A. L., Hoch, J. A. & Losick, R. (Am. Soc.Microbiol., Washington, DC), pp. 483–517.

3. Molle, V., Fujita, M., Jensen, S. T., Eichenberger, P., Gonzalez-Pastor, J. E., Liu, J. S. & Losick, R. (2003) Mol. Microbiol. 50, 1683–16701.

4. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–402.

5. Letunic, I., Copley, R. R., Schmidt, S., Ciccarelli, F. D., Doerks, T., Schultz, J., Ponting, C. P. & Bork, P. (2004) Nucleic Acids Res. 32, D142–D144.

6. Ogura, M., Shimane, K., Asai, K., Ogasawara, N. & Tanaka, T. (2003) Mol. Microbiol. 49, 1685–1697.

Table 5. Comparison of transfer frequencies using different mating protocols

*Ratio of donor (JMA168) to recipient (CAL419) cells.

^†Agar used during 3-hr incubation at 37°C on filter.

^‡Frequency, mean number of transconjugants per donor (±SEM).

Table 6. rap and phr genes in Bacillus mobile genetic elements

1. Takemaru, K., Mizuno, M., Sato, T., Takeuchi, M. & Kobayashi, Y. (1995) Microbiology 141, 323–327.

2. Meijer, W. J., Wisman, G. B., Terpstra, P., Thorsted, P. B., Thomas, C. M., Holsappel, S., Venema, G. & Bron, S. (1998) FEMS Microbiol. Rev. 21, 337–68.

3. Gleave, A. P., Mountain, A. & Thomas, C. M. (1990) J. Gen. Microbiol. 136, 905–912.

4. Dhaese, P., Seurinck, J., De Smet, B. & Van Montagu, M. (1985) Nucleic Acids Res. 13, 5441–5455.

5. Koehler, T. M. & Thorne, C. B. (1987) J. Bacteriol. 169, 5271–5278.

6. Read, T. D., Peterson, S. N., Tourasse, N., Baillie, L. W., Paulsen, I. T., Nelson, K. E., Tettelin, H., Fouts, D. E., Eisen, J. A., Gill, S. R., et al. (2003) Nature 423, 81–86.

7. Parini, C., Guglielmetti, S., Mora, D. & Ricci, G. (2004) Plasmid 51, 192–202.

8. Rasko, D. A., Ravel, J., Okstad, O. A., Helgason, E., Cer, R. Z., Jiang, L., Shores, K. A., Fouts, D. E., Tourasse, N. J., Angiuoli, S. V., et al. (2004) Nucleic Acids Res. 32, 977–988.

Supporting Text

Changes in gene expression caused by overproduction of RapI. We did two types of experiments to evaluate changes in gene-specific mRNA levels caused by overexpression of rapI (Table 4). Type I was analyzed on DNA microarrays containing PCR products of virtually all the Bacillus subtilis ORFs. Type II was analyzed on DNA microarrays containing a unique oligonucleotide for virtually every ORF.

In type I experiments, RNA was harvested from cells containing the LacI-repressible, isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible fusion Pspank(hy)-rapI (strain JMA28) grown without IPTG (no overexpression) or 30 min after induction with IPTG. Fluorescently labeled cDNA prepared from these samples was cohybridized to PCR arrays containing DNA amplified from >99% of the B. subtilis ORFs.

In type II experiments, RNA was harvested from Pspank(hy)-rapI cells and from control cells [Pspank(hy), JMA35, no insert downstream from Pspank(hy)]. Fluorescently labeled cDNA was prepared from these samples, mixed with a labeled reference sample, and hybridized to arrays containing 65-mer oligonucleotides complementary to all the annotated B. subtilis ORFs.

Both experiments were performed with three independent sets of cultures. Many genes in the integrative and conjugative element ICEBs1 appeared to have much greater overexpression in type II, compared with type I, experiments (Table 4). Much of this overexpression is likely because of the control sample used for normalization. In the type I experiments, RNA levels in rapI-overexpressing cells were compared with uninduced Pspank(hy)-rapI cells, which have a higher level of ICEBs1 gene expression and excision than do control Pspank(hy) cells because of incomplete repression of the Pspank(hy)-rapI promoter in the absence of inducer (data not shown). However, in the type II experiments, RNA levels in rapI-overexpressing cells were compared with Pspank(hy) cells, which do not have increased levels of ICEBs1 gene expression and excision.

ICEBs1-transfer frequency depends on donor-to-recipient ratio and growth medium. We observed a large range of transfer efficiencies of ICEBs1, depending on the specific mating conditions (compare mating data in Table 1 with Table 2). Under a given set of conditions, mating frequencies were quite consistent. However, when two different sets of mating conditions in two different types of experiments were compared, the differences in transfer frequencies were significant. For example, under one set of conditions, transfer of ICEBs1 from Δ (rapI phrI) donor cells into ICEBs1⁺ comK recipients was not detected (<3 × 10^–8 transconjugants per donor) but occurred at a frequency of ≈1 × 10^–5 per donor cell under a different set of conditions.

There were many differences between the experiments that gave the various mating frequencies, including different donor and recipient strains, different growth media, differences in the amount of time the donor and recipient were together before filter mating, and differences in the donor-to-recipient ratio. To explore what contributed to the significant differences in mating frequencies, we tested many of these parameters in side-by-side comparisons. We used strain JMA168 [ICEBs1 Δ(rapI phrI)::kan amyE::(Pspank(hy)-rapI)] as a donor and strain CAL419 (ICEBs1⁰ comK::cat str) as a recipient. Excision of ICEBsI in the donor was induced by the addition of IPTG to overexpress rapI, and cells were mixed 1 h later. Different mating conditions were tested in parallel, and transconjugants were selected for resistance to kanamycin (from ICEBs1) and streptomycin (from recipient).

We found that the mating frequency was affected by both the donor-to-recipient ratio and the medium used for the filter mating. Transfer increased ≈10-fold in filter matings performed with a ratio of ≈1 donor cell to 100 recipient cells, relative to filter matings performed with a ratio of ≈1 donor cell to 1 recipient cell (Table 5). Donor-to-recipient ratios of ≈1:10, ≈1:25, ≈1:200, and ≈1:400 gave transfer frequencies similar to the ≈1:100 ratio (data not shown). These results indicate that the availability of recipient cells likely limits the frequency of mating from donor to recipient cells when an equal number of donor and recipient cells are present.

We also observed an ≈100-fold increase in transfer efficiency when matings of ≈1 JMA168 donor to 100 CAL419 recipients were performed on nutrient broth (Difco) sporulation agar (DSM), compared with matings performed on LB agar (Table 5). This increase in transfer on DSM could be caused by the presence of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺) or by the physiological effects of nutritional differences between DSM and LB agar. Taken together, these experiments demonstrate that factors in addition to PhrI peptide signaling affect the efficiency of ICEBs1 transfer.

Of note is that matings done on DSM at a ratio of ≈1 donor to 100 recipients gave rise to multiple transconjugants per donor. It is most likely that a single donor is mating with multiple recipients. It is also possible that transconjugants serve as donors during the course of the experiment.

Supporting Methods

Strain information. Strains used are listed in Table 3, and the construction of specific alleles not described in the main text is described below. Null mutations generated by double-crossover recombination of alleles into the chromosome were verified by PCR.

Generation of spontaneous streptomycin-resistant mutants. The B. subtilis str strain (CAL84) and the Bacillus licheniformis str strain (REM42) were generated by selecting for spontaneous streptomycin resistance of the parental strains JH642 and ATCC11946, respectively, on LB plates containing streptomycin (100 μg/ml). CAL84 and REM42 are resistant to streptomycin and sensitive to spectinomycin. The str allele from CAL84 was used to generate strains CAL88 (comK::spc str), CAL89 (ICEBs1⁰ comK::spc str), and CAL419 (ICEBs1⁰ comK::cat str).

Generation of an ICEBs1-cured (ICEBs1⁰) strain. A strain cured of ICEBs1 (JMA222) was generated by growing ΔimmR208::cat cells (immR encodes a repressor of ICEBs1 gene expression) in the absence of antibiotic selection for many generations. The immR mutant has an increased frequency of ICEBs1 excision, and after many generations of growth without selection, 9 of 100 colonies from LB agar plates were sensitive to chloramphenicol, indicating that these cells had lost the immR208::cat allele. One isolate, JMA222, was chosen for further study.

The absence of ICEBs1 at the attachment (att) site was confirmed through PCR using primers (oJMA93 and oJMA100) that amplify across the unoccupied att site. Sequencing of this PCR product revealed that it contains a single att site surrounded by the chromosomal sequence that normally flanks the integrated ICEBs1. This same unoccupied att-site structure is observed in sequenced PCR products from cells in which rapI overexpression has stimulated excision of ICEBs1. In addition, by using ICEBs1-specific primers, we were unable to detect the element elsewhere in the genome, nor were we able to detect any of the ICEBs1 genes by using DNA microarrays (data not shown). Based on these data, we believe that ICEBs1, excised through the normal excision mechanism in JMA222, failed to reintegrate and was lost from progeny cells during growth and cell division.

We also found that ICEBs1 was missing in some lab strains of B. subtilis. We tested for the presence of ICEBs1 by using PCR to detect int, immR, and rapI phrI. Sequences of the primers used to amplify these regions are listed below. We also tested for insertion of ICEBs1 at attB (the chromosomal att site in tRNS-leu2) by detecting the region spanning attR (primers oJMA97 and oJMA100, listed below). In addition, we tested for the unoccupied attB site (repaired chromosomal junction), as described. Presence of the unoccupied attB site and absence of the region spanning attR indicates that, if ICEBs1 is present, it is not integrated at attB. We found that, in addition to the lab strain JH642, strains 168 (1), CRK6000 (2), and NCIB3610 (3) all contained ICEBs1 integrated at attB. PCR assays indicated that strains PY79 (4) and YB886 (5) likely do not contain ICEBs1, because the individual regions containing attR, int, immR, and rapI phrI were undetectable, and the attB site was unoccupied (data not shown). It is possible that these strains have a form of ICEBs1 elsewhere in the genome, which has enough sequence divergence that it is not recognized by the primers used for amplification; however, genomic DNA microarrays comparing DNA content between JH642 and YB886 failed to detect any ICEBs1 genes (data not shown), indicating that, if present, ICEBs1 contains significantly divergent sequences in all of its genes.

Δ(ICEBs1)206::cat.Bs1206catBs1attRcat

ICEBs1::kan. ICEBs1::kan is functionally ICEBs1⁺ and contains the kanamycin-resistance gene from pGK67 (7) inserted between the 3' end of yddM and attR.

Deletion–insertion of rapI and phrI. The Δ(rapI phrI)342::kan insertion–deletion was generated by replacing the 3' end of rapI and all of phrI with the kanamycin-resistance gene in pGK67 (7).

Null mutations in int and immR. Δint205::cat and ΔimmR208::cat were generated by replacing int or immR with the chloramphenicol-resistance gene from pGEM-cat (6).

Preparation of DNA Microarrays. PCR products were resuspended in 50% DMSO and spotted onto Corning GAPS II slides. Oligonucleotides were resuspended in 50% DMSO at a concentration of 25 μM and spotted onto Corning UltraGAPS slides. The slides were stored at room temperature until use. The PCR product arrays stored well for at least 2 years. The oligonucleotide arrays stored well for at least 6 months.

Before hybridization with biological samples, DNA was crosslinked to the glass slides by using a UV Stratalinker (Stratagene) at 90 mJ for the PCR product arrays and 600 mJ for the oligonucleotide arrays. After crosslinking, arrays were incubated in prehybridization buffer [5× SSC (0.75 M sodium chloride/75 mM sodium citrate, pH 7.0/1% SDS/1% BSA)] for at least 45 min at 42°C. Prehybridized slides were washed in double-distilled water. Excess water was removed by centrifugation and drying with nitrogen gas.

Reverse transcription and labeling of RNA for microarray experiments. RNA (10 µg) from each sample was reverse-transcribed with Superscript II reverse transcriptase (Invitrogen) in the presence of aminoallyl-dUTP (Sigma or Ambion). RNA samples were combined with 2.5 μg random hexamers (Operon or Qiagen) and incubated at 70°C for 10 min, followed by incubation at 4°C for 5 min. Reverse-transcription reactions (30 µl) were started by adding a mix containing additional reaction components to make the final reaction conditions: 1× RT buffer (Invitrogen), 10 mM DTT (Invitrogen), 300 units of RT, 0.5 mM dATP, dCTP, and dGTP (Invitrogen), 0.1 mM dTTP (Invitrogen), 0.4 mM aminoallyl-dUTP, and 20 units of RNase Out (Invitrogen). The reverse-transcription reactions were incubated at 25°C for 10 min, at 42°C for 70 min, and then shifted to 70°C for 15 min to stop reactions. RNA in the reactions was degraded by adding sodium hydroxide (33 mM final concentration) and incubating at 70°C for 10 min. Hydrogen chloride (25 mM final concentration) was added to neutralize the reactions.

Labeled cDNAs were purified with either Qiagen MinElute or QIAquick PCR purification columns according to the manufacturer’s protocol, with the exception that the columns were washed with 75% ethanol instead of Buffer PE and were eluted with sterile H₂O. Samples purified on Qiagen QIAquick PCR purification columns were dried by centrifugation under vacuum and resuspended in a smaller volume of sterile water. Sodium bicarbonate (pH 9) was added to each sample to adjust the pH before coupling. To couple the fluorescent dyes to aminoallyl-modified uracil in the cDNA, the amine-reactive Cy5 and Cy3 dyes (Amersham Pharmacia) were added to the cDNA and incubated for 1 h in the dark, mixing every 15 min. Coupling reactions were quenched by incubation with hydroxylamine (1.125 M final concentration) for 15 min in the dark.

PCR primer sequences. The following primers (5' to 3') were used to assay excision of ICEBs1: Chromosomal junction formed after excision of ICEBs1, oJMA93-GACGAA-TATGGCAAGCCTATGTTAC and oJMA100-GGGTATACAATCATGGGTGATC-GAG; ICEBs1 circular intermediate, oJMA95-CTGGACTAAGATGTGGTGAAA-TGCTC and oJMA97-CTGTAAATTATGAATCTCAGATTGTTAATCCTGC; cotF region as control, oLIN93-GCAGCGGCGTTCTGCAAGC and oLIN94-CACTTAG-TCACCTCGTATCATC; amyE::Pspank(hy) region as control for cells in mixed culture, oJMA177-CTACCGAGATATCCGCACCAACGC and oJMA178-CTCTGACCAG-ACACCCATCAACAG.

The following primers (5' to 3') were used to detect ICEBs1. Underlined sequences contain added restriction sites and extra nucleotides that are not complementary to ICEBs1 sequence. Primer internal to ICEBs1, upstream of attR, oJMA97-CTGTAAAT-TATGAATCTCAGATTGTTAATCCTGC; Primers to amplify int, oJMA127-ATATGCTAGCGCCCACAAACTGCCCACTTACC and oJMA128-ATATGTCGAC-CAGAATCTATTCACACGAAATAAGCGC; Primers to amplify immR, oJMA122-ATATAAGCTTCTCTCCATAAAGAAGAAACAAACACTCC and oJMA123-CAGAGCTAGCGTTATCACTCTTTCTTCTTTAATTCGTCAATG; Primers to amplify rapIphrI, oJMA25-ATAATTGTCGACCGCACAATTTTATGTAAG and oJMA64-ATCTACGCATGCTTCCAATTATCTAAGCTATG.

PCR conditions. Each reaction (50 μl) contained primers at a final concentration of 1 µM, 200 µM dNTPs, 1× Taq Buffer (Roche), and 1.25 units of Taq DNA polymerase (Roche). For nonlinear-range PCR, reactions were amplified for 3 min at 94°C, followed by 30 cycles of 30 s at 94°C, 60 s at 56°C, and 2 min at 72°C. These cycles were followed by a 5-min extension at 72°C. For linear-range (quantitative) PCR, reaction conditions were the same, except that the number of cycles was reduced to 26.

1. Spizizen, J. (1958) Proc. Natl. Acad. Sci. USA 44, 407–408.

2. Moriya, S., Kato, K., Yoshikawa, H., & Ogasawara, N. (1990) EMBO J. 9, 2905–2910.

3. Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., & Kolter, R. (2001) Proc. Natl. Acad. Sci. USA 98, 11621–11626.

4. Youngman, P., Perkins, J. B., & Losick, R. (1984) Plasmid 12, 1–9.

5. Yasbin, R. E., Fields, P. I., & Andersen, B. J. (1980) Gene 12, 155–9.

6. Youngman, P., Poth, H., Green, B., York, K., Olmedo, G., & Smith, K. (1989) in Regulation of Procaryotic Development, eds. Smith, I., Slepecky, R. A., & Setlow, P. (Am. Soc. Microbiol., Washington, DC), pp. 65–87.

7. Lemon, K. P., Kurtser, I., & Grossman, A. D. (2001) Proc. Natl. Acad. Sci. USA 98, 212–217.

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