A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987

Abstract

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

Figure 1

Gene rearrangement of Bacillus cereus gerE. (a) Schematic of gene rearrangements of B. subtilis sigK and B. cereus gerE. The composite sigK and gerE genes after the rearrangement encode a sporulation-specific sigma factor, σ^K, and a transcriptional factor, GerE, respectively, which govern mother cell-specific gene expression at the late stage of sporulation. RNAP, RNA polymerase. (b) Whole nucleotide sequence of gerE from Bacillus cereus ATCC10987. Deduced amino acid sequence is shown under the nucleotide sequence. The gin element is inserted at position 138–146 nt (indicated by the red box). Nucleotides shaded green and purple correspond to the coding regions of 5′-gerE and gerE-3′, respectively. START, start codon; STOP, stop codon.

Figure 2

Genetic organization of the gin element and disrupted gerE. (a) The genetic map of the gin element is shown. Red, serine recombinase genes; blue, restriction-modification system genes; yellow, recJ. (b) Comparative genetic organization of gin elements in Bacillus cereus and B. toyonensis. The gin elements from B. cereus strains ATCC10987, FT9, AH820, and Q1 and from B. toyonensis BCT-7112 are shown. The size (kb) of the gin element is shown to the right.

Figure 3

Verification of gerE rearrangement during sporulation in B. cereus ATCC10987. (a) Schematic showing the gerE locus before and after the rearrangement. H, HindIII restriction sites; triangles, DNA recombination sites; arrows, primers for PCR; thick lines, DNA probes for Southern blotting. (b) Cell morphologies of B. cereus vegetative and sporulating cells. B. cereus cells at T_-1 (vegetative phase) and T₆ (sporulation phase) were observed using phase contrast (left panel) and fluorescent microscopy (right panel). T₀ was defined as the onset of the sporulation. The fluorescence images were generated by merging pictures of membrane staining with FM4-64 (red) and DNA staining with SYTO16 (green). Images from T₋₁ to T₁₂ are shown in Supplementary Figure S3. (c) PCR detection of gerE rearrangement. Chromosomal DNA was isolated from B. cereus vegetative (T₋₁; veg) and sporulating (T₆; spo) cells. The junction sites of the composite gerE (attB; 925 bp) and gin element (attG; 690 bp) were detected by PCR using the primers indicated in (a). (d) Insensitivity to mitomycin C (MMC). B. cereus cells were cultured at 37 °C in LB medium or sporulation medium (DSM). Vegetative cells (OD₆₀₀ = 0.5) in the LB medium were treated with (+) or without (−) 0.5 μg/ml MMC for 90 min. The DSM culture was collected at T₋₁ (veg) and T₆ (spo). The excised gin element was detected by PCR with the gin-specific primers. (e) Southern blotting. Chromosomal DNA from sporulating cells at various time points during sporulation was digested with HindIII, and subjected to Southern blotting using the gerE-specific (top panel) and the gin-specific (bottom panel) probes. The original gel and blot images of Fig. 3 are presented in Supplementary Figure S7.

Figure 4

Nucleotide sequences of the DNA recombination sites. (a) DNA recombination sites before and after the gerE rearrangement were amplified by PCR and analyzed using a DNA-sequencer. The 9 bp consensus sequence between the DNA recombination sites is shown in the red box. (b) Nucleotide sequences at the DNA recombination sites before and after the rearrangement. The deduced amino-acids sequences of GerE are shown above the nucleotide sequence. The consensus sequence among the four att sites is indicated by the red box and the inverted repeat sequences are denoted by arrows. (c) Alignment of the amino acid sequences of the B. cereus and B. subtilis GerE proteins. The amino acid sequences encoded by the B. cereus truncated and composite gerE and B. subtilis gerE are shown as 5′-GerE, 3′-GerE, GerE_Bc, and GerE_Bs, respectively. The boxed amino acid sequences correspond to the region encoded by the 9 bp consensus sequence. Asterisks indicate the non conserved amino acids in B. cereus and B. subtilis.

Figure 5

Gene complementation test for gerE. The B. cereus truncated (5′-gerE) and composite gerE (gerE _Bc) were introduced into the amyE locus in the chromosome of the B. subtilis gerE-deletion mutant strain, GEd. (a) lacZ expression under control of the cotG promoter. A P_cotG –lacZ construct was introduced into B. subtilis 168 (wild type; filled circle), GEd (ΔgerE _Bs; filled triangle), GEd-5 (ΔgerE _Bs, 5′-gerE ⁺; open circle), and GEd-C (ΔgerE _Bs, gerE _Bc ⁺; open triangle). The B. subtilis strains harboring the P_cotG –lacZ construct were induced to sporulate by cultivation in DSM at 37 °C. LacZ activity was measured at the indicated time points. (b) Germination rates. Spores from B. subtilis 168 (wild type; fill circle), GEd (filled triangle), GEd-5 (open circle), and GEd-C (open triangle) were treated at 70 °C for 30 min, and subsequently 1 mM L-alanine was added to induce germination. Relative OD₆₀₀ values at each time point to the initial OD₆₀₀ are plotted. Decrease in the OD₆₀₀ reflects the initiation of germination. (c) Lysozyme sensitivity. Spores prepared from B. subtilis 168 (filled circle), GEd (filled triangle), GEd-5 (open circle), and GEd-C (open triangle) were treated with lysozyme at 37 °C. Relative OD₆₀₀ values at each time point to the initial OD₆₀₀ are plotted. A decrease in the OD₆₀₀ value indicates the spore’s sensitivity to lysozyme. Error bars indicate the standard deviations based on three independent experiments.

Figure 6

Identification of gin-encoded genes required for gerE rearrangement. (a) Schematic of the deletion series of the gin element. The deleted regions within the gin element in B. subtilis 168Gin are shown. Arrows indicate the PCR primers used to detect gerE rearrangement. The location of the putative RDF for GirC was inferred as the shaded area. (b) Detection of gerE rearrangement. PCR was performed to detect the composite gerE (925 bp) after rearrangement in 168Gin, GCd, GABd, and GR1–3 strains using chromosomal DNA from cells at the vegetative (T₋₁) and sporulation (T₆) phases. (c) Conservation of girX in the gin elements in B. cereus and B. toyonensis strains. The girC and girX genes are colored red and orange, respectively. (d) Alignment of the amino acids sequences of GirX from B. cereus strains ATCC10987, AH820, and Q1, and from B. toyonensis BCT-7112. The amino acids conserved in three out of the four and in all four are shown in grey and black, respectively. (e) Complementation test for GirX. The girX gene was introduced into the GR2 strain (GR2X). PCR was performed to detect the composite gerE after the rearrangement under the same conditions as shown in (b). The original agarose gel images of Fig. 6 are presented in Supplementary Figure S7.

Figure 7

Transcription of girC, girX, and gerE _Bc in B. cereus during sporulation. (a) Diagram of the primer positions for the reverse transcription and PCR reactions. (b) Detection of the transcripts from the girC, girX, and the composite gerE _Bc gene. B. cereus cells were cultured at 37 °C in sporulation medium (DSM). Total RNA was extracted from the cells at various times during sporulation. cDNA was obtained by a reaction using reverse transcriptase with the girC-, girX-, and gerE _Bc-specific primers, and amplified by 18 cycles of PCR with the appropriate primer sets shown in (a). The PCR products were separated by 2% agarose gel electrophoresis. The original agarose gel images are presented in Supplementary Figure S7.

Figure 8

In vitro recombination assay. (a) Diagram of the in vitro recombination reactions. The in vitro recombination assays were performed using plasmid DNA carrying the att sites as the substrates for integration (pMDTIn) and excision (pMDTEx). (b) Purified GirC (1 μg) and GirX (1 μg) proteins fused with the his₆-tag at their C-termini were loaded into SDS-PAGE (12%) and Tricine-SDS-PAGE (12%) gels, respectively. The original gel images are presented in Supplementary Figure S7. (c) Integration reaction. The DNA substrate, pMDTIn (200 ng; attB + attG [supercoiled]), was reacted with GirC-His₆ (0–1.5 μM) at 37 °C for 1 hr. The recombination products, attL and attR, were analyzed by agarose gel electrophoresis. (d) Excision reaction. The DNA substrate, pMDTEx (200 ng; attL + attR), was reacted with GirC (0 or 1 μM) and GirX (0–4 μM) at 37 °C for 1 hr. The recombination products were analyzed by agarose gel electrophoresis. (e) Inhibition of integration by GirX. The integrative substrate, pMDTIn was incubated at 37 °C for 1 hr in the absence (−) or the presence of GirC (+; 1 μM) and GirX (+; 4 μM). The recombination products were analyzed by agarose gel electrophoresis. (f) In vitro recombination between B. subtilis gerE (attB _Bs) and attG. The attB site of pMDTIn was replaced with attB _Bs to generate pMDTIn-Bs. pMDTIn-Bs was reacted with 1 μM GirC at 37 °C for 1 hr. Arrowheads indicate the recombination products, attL and attR. The original agarose gel images of Fig. 8 are presented in Supplementary Figure S7.