Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis

Abstract

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σ^H factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. β-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.

Keywords: Bacillus thuringiensis; disporic septum; spoVS; sporulation; σH.

FIG 1

Basic description of spoVS genes in B. thuringiensis HD73. (A) Map of the RS20185–RS20195 locus in the B. thuringiensis HD73 genome. The deleted region represents the fragment deleted in the spoVS1 null mutant. P_spoVS1 is the promoter region used in the β-galactosidase activity assay. The bent arrow represents the transcription start site. ORFs are indicated by large open arrows. The scale bar corresponds to 200 bp. (B) Map of the RS12220–RS12230 locus in the B. thuringiensis HD73 genome. The deleted region represents the fragment deleted in the spoVS2 null mutant. P_spoVS2 is the promoter region used in the β-galactosidase activity assay. The bent arrow represents the transcription start site. ORFs are indicated by large open arrows. The scale bar corresponds to 200 bp. (C) Comparison of the amino acid sequences of three SpoVS proteins from B. thuringiensis HD73 and B. subtilis PY79. Sequence alignment of SpoVS1 in B. thuringiensis HD73 and SpoVS in B. subtilis PY79 reveals 92% identity between the two proteins. Sequence alignment of SpoVS2 in B. thuringiensis HD73 and SpoVS in B. subtilis PY79 reveals 76% identity between the two proteins. The amino acid identity between SpoVS1 and SpoVS2 is 72%.

FIG 2

Transcriptional regulation of the spoVS1 and spoVS2 genes. (A) Effects of σ^H on P_spoVS1 and P_spoVS2 expression. β-Galactosidase activity was assessed in the wild-type B. thuringiensis HD73 (squares) and the sigH null mutant strains (circles) containing the plasmid-borne transcriptional fusion P_spoVS1-lacZ (red symbols) or P_spoVS2-lacZ (black symbols). The bacteria were grown at 30°C in SSM medium, and samples were taken at the indicated time points. Time zero (T₀) is the end of the exponential growth phase, and T_n is n hours after T₀. Each data point represents the mean value from at least three independent replicates. Error bars show standard errors of the means. (B) Electrophoretic mobility shift assay (EMSA) for detecting protein-DNA interactions using FAM-labeled P_cry1Ac and increasing concentrations of recombinant SigH-His. The lanes contained 0, 50, 100, 200, and 300 ng/μl of SigH-His. (C) EMSA for detecting protein-DNA interactions using FAM-labeled P_spoVS1 and increasing concentrations of recombinant SigH-His. The lanes contained 0, 50, 80, 120, 160, and 180 ng/μl of SigH-His. (D) EMSA for detecting protein-DNA interactions using FAM-labeled P_spoVS2 and increasing concentrations of recombinant SigH-His. The lanes contained 0, 30, 90, 60, and 200 ng/μl of SigH-His.

FIG 3

Observations of the sporulation process using a laser scanning confocal microscope. The polar septa and engulfment of B. thuringiensis HD73 (wild-type strain), HD(ΔspoVS1), HD(ΔspoVS2), HD(ΔspoVS1ΔspoVS2), HD(ΔspoVS1ΩspoVS1), HD(ΔspoVS2ΩspoVS2), HD(ΔspoVS1ΩspoVS2), and HD(ΔspoVS2ΩspoVS1) were observed using a laser scanning confocal microscope at T₂, T₃/T₈/T₁₂, and T₈/T₁₂/T₁₅ after incubation in SSM at 30°C with shaking at 220 rpm. Cell membrane is visible as red fluorescence. Red lines represent membranes stained with FM 4-64, and green lines represent forespores stained with MitoTracker green FM (MTG). Yellow arrows indicate straight polar septa. White arrows indicate curved polar septa. Blue arrows indicate forespores. Scale bars = 5 μm.

FIG 4

Observations of mother spore release under optical microscopy. (A) Spore release of HD73 (wild-type strain), HD(ΔspoVS1), HD(ΔspoVS1ΔspoVS2), HD(ΔspoVS1ΩspoVS2), HD(ΔspoVS1ΩspoVS1), and HD(ΔspoVS2ΩspoVS1). Observations were made using optical microscopy at T₂₄, T₄₈, and T₇₂ after incubation in SSM at 30°C with shaking at 220 rpm. Scale bars = 10 μm. (B) Spore release of HD73 (wild-type strain) and HD(ΔspoVS2) mutant. Observations were made using optical microscopy at T₂₄, T₂₈, and T₃₂ after incubation in SSM at 30°C with shaking at 220 rpm. Scale bars = 10 μm.

FIG 5

Comparison of the sporulation frequencies of HD73 (wild-type strain), HD(ΔspoVS2), HD(ΔspoVS1), HD(ΔspoVS1ΔspoVS2), HD(ΔspoVS2ΩspoVS2), HD(ΔspoVS1ΩspoVS1), HD(ΔspoVS2ΩspoVS1), and HD(ΔspoVS1ΩspoVS2). (A) The counts of total cells and spores in HD73, HD(ΔspoVS1), HD(ΔspoVS2), HD(ΔspoVS1ΔspoVS2), HD(ΔspoVS2ΩspoVS2), HD(ΔspoVS1ΩspoVS1), HD(ΔspoVS2ΩspoVS1), and HD(ΔspoVS1ΩspoVS2). Deletion of the spoVS1 gene reduced the numbers of cells and spores, but deletion of the spoVS2 gene did not impact the numbers of cells and spores. Self-complement of spoVS1 reduced the numbers of cells and spores, but the complement of the spoVS2 gene to the HD(ΔspoVS1) mutant could restore the total numbers of cells and spores. Each bar represents the mean value from at least three independent replicates. The error bars represent standard deviations. (B) The sporulation frequencies of all strains whose total cell counts and spore counts are shown in panel A. The sporulation frequency was defined as the ratio of the number of spores to the total number of cells, multiplied by 100. The percentage represents the average sporulation frequency. The sporulation frequency of every spoVS-related strain was compared with that of the wild-type strain (HD73), and the data were analyzed with SPSS (version 19.0) using the t test (*, P ≤ 0.05; **, P ≤ 0.01). The error bars represent standard deviations.

FIG 6

Effects of various mutations on pro-sigE and spoIIE expression. (A) Disporic septa of HD(ΔsigE) mutant (a), HD(ΔspoVS1) mutant (b), HD(ΔspoVS1ΔspoVS2) double mutant (c), and HD(ΔspoVS1ΩspoVS2) mutant (d) were observed using a laser scanning confocal microscope at T₄ after incubation in SSM at 30°C with shaking at 220 rpm. Cell membranes are visible as red fluorescence. Red lines represent membranes stained with FM 4-64. Blue arrows indicate disporic septa. Scale bars = 7.5 μm. (B) β-Galactosidase activities were assessed for HD73 (wild-type) (black circles), HD(ΔspoVS1) mutant (red squares), HD(ΔspoVS2) mutant (green triangles), and HD(ΔspoVS1ΔspoVS2) mutant (blue rhombuses) containing the plasmid-borne transcriptional fusion P_pro-sigE-lacZ. The bacteria were grown at 30°C in SSM medium, and samples were taken at the indicated time points. T₀ is the end of the exponential growth phase, and T_n is n hours after T₀. Each data point represents the mean value from at least three independent replicates. Error bars show the standard errors of the means. (C) β-Galactosidase activity was assessed for HD73 (wild-type) (black circles), HD(ΔspoVS1) mutant (red squares), HD(ΔspoVS2) mutant (green triangles), and HD(ΔspoVS1ΔspoVS2) mutant (blue rhombuses) containing the plasmid-borne transcriptional fusion P_spoIIE-lacZ. The bacteria were grown at 30°C in SSM medium, and samples were taken at the indicated time points. T₀ is the end of the exponential growth phase, and T_n is n hours after T₀. Each data point represents the mean value from at least three independent replicates. Error bars show the standard errors of the means.