SpoVG is Necessary for Sporulation in Bacillus anthracis

Abstract

The Bacillus anthracis spore constitutes the infectious form of the bacterium, and sporulation is an important process in the organism's life cycle. Herein, we show that disruption of SpoVG resulted in defective B. anthracis sporulation. Confocal microscopy demonstrated that a ΔspoVG mutant could not form an asymmetric septum, the first morphological change observed during sporulation. Moreover, levels of spoIIE mRNA were reduced in the spoVG mutant, as demonstrated using β-galactosidase activity assays. The effects on sporulation of the ΔspoVG mutation differed in B. anthracis from those in B. subtilis because of the redundant functions of SpoVG and SpoIIB in B. subtilis. SpoVG is highly conserved between B. anthracis and B. subtilis. Conversely, BA4688 (the protein tentatively assigned as SpoIIB in B. anthracis) and B. subtilis SpoIIB (SpoIIB_Bs) share only 27.9% sequence identity. On complementation of the B. anthracis ΔspoVG strain with spoIIB_Bs, the resulting strain pBspoIIB_Bs/ΔspoVG could not form resistant spores, but partially completed the prespore engulfment stage. In agreement with this finding, mRNA levels of the prespore engulfment gene spoIIM were significantly increased in strain pBspoIIB_Bs/ΔspoVG compared with the ΔspoVG strain. Transcription of the coat development gene cotE was similar in the pBspoIIB_Bs/ΔspoVG and ΔspoVG strains. Thus, unlike in B. subtilis, SpoVG appears to be required for sporulation in B. anthracis, which provides further insight into the sporulation mechanisms of this pathogen.

Keywords: Bacillus anthracis; spoIIB; spoVG; sporulation.

Figure 1

Schematic of construction of the B. anthracis ΔspoVG mutant and complemented strains. (a) Construction of the ΔspoVG strain. The construction method has been described in detail previously [24]. Briefly, the recombinant allelic exchange vector pKMUSD containing an I-SceI site was constructed. Then, pKMUSD was introduced into B. anthracis strain A16R, followed by introduction of pSS4332. Expression of the endonuclease I-SceI from pSS4332 promotes homologous recombination. When homologous recombination is completed, pSS4332 is driven out, producing a ∆spoVG mutant. (b) The complementation plasmids pBE2AspoVG and pBE2spoIIB_Bs were constructed as described in Materials and Methods. These plasmids were introduced into B. anthracis A16RΔspoVG competent cells, to yield strains RΔspoVG and pBspoIIB_Bs/ΔspoVG, respectively.

Figure 2

Role of SpoVG in sporulation. (a) Sporulation efficiency of B. anthracis strains A16R (wild-type), ΔspoVG and RΔspoVG (complemented strain) cultured in DSM for 24 h. Values shown are means ± standard deviations (SDs) of triplicate experiments. (b) A16R, ΔspoVG and RΔspoVG strain cultures (24 h) were stained with malachite green and safranin O. Spores and vegetative cells are stained green and red, respectively. Scale bar, 10 μm. (c) Ultrastructural observations of the three strains using transmission electron microscopy. Red double arrows indicate spores. Scale bar, 2 μm.

Figure 3

Defects in the formation of asymmetric septum in the B. anthracis ΔspoVG strain are associated with low transcriptional levels of spoIIE. (a) Confocal laser-scanning micrograph (scale bar, 10 µm) of the A16R (wild-type), ΔspoVG, and RΔspoVG strains at T₁, T₃, and T₁₇ (37 °C), T_n being n hours after T₀ (the end of the exponential growth phase). The cell membrane is visible as red fluorescence. Yellow, white and red arrows indicate asymmetric septum, engulfed cells (prespores), and mature spores, respectively. (b) Relative mRNA expression of spoIIE determined in the ΔspoVG and RΔspoVG strains compared with A16R (wild-type) cells at T₁ using RT-qPCR. Values represent means ± SDs of triplicate experiments. (c) Transcription of P_spoIIE–lacZ in A16R (wild-type) cells (green line) and ΔspoVG-mutant cells (red line) grown in DSM. Values represent the means of at least three independent replicates; error bars represent SDs.

Figure 4

Amino acid sequence similarity of SpoVG and SpoIIB from species in the B. cereus group compared with those in B. subtilis. (a) Phylogenetic trees based on amino acid sequence alignment of SpoVG and SpoIIB, respectively. The unweighted pair group method with mean averages (UPGMA) tree was based on alignment of 10 amino acid sequences of SpoVG and SpoIIB proteins from strains belonging to the B. cereus group (seven sequences, including sequences from B. anthracis), B. subtilis (two sequences), and B. amyloliquefaciens (one sequence) available in the NCBI database (https://www.ncbi.nlm.nih.gov/). Multiple sequence alignment was conducted using ClustalX, and the tree was generated using Mega X software. The schematic shows regions of similarity rather than the complete sequence because of the length of the SpoIIB protein. SpoVG is highly conserved between B. anthracis and B. subtilis, while BA4688 (from B. anthracis) and SpoIIB_Bs (from B. subtilis) share a low amino acid sequence similarity. (b) Comparison of the spoIIB locus across Bacillus species. Arrows indicate the orientations of open reading frames. Original genome annotations are listed, and the names of organisms are abbreviated to: BA, B. anthracis Ames, and BS, B. subtilis 168. BA4688 represents the putative protein in B. anthracis that has the highest similarity to B. subtilis SpoIIB, but it still shares only a small region of similarity with the latter protein. There is some difference in the genetic location of BA4688 and spoIIB_Bs between B. anthracis and B. subtilis.

Figure 5

Sporulation of the B. anthracis pBspoIIB_Bs/ΔspoVG strain. (a) Strains were cultured in DSM for 24, 72, and 120 h. The cultures were serially diluted and 10 μL aliquots of each dilution (10⁻¹ through 10⁻⁵) were plated on LB-agar. After heat inactivation, spores were diluted and plated in the same manner. Images of plates after overnight incubation at 37 °C are shown. The ΔspoVG and pBspoIIB_Bs/ΔspoVG strains did not form heat-resistant spores. (b) Cultures of four strains were stained with malachite green and safranin O at 120 h. Spores and vegetative cells were stained green and red, respectively. Scale bar, 10 μm.

Figure 6

Morphological and molecular characteristics of the B. anthracis pBspoIIB_Bs/ΔspoVG strain during sporulation. (a) Confocal laser-scanning micrographs (scale bar, 10 µm) of strain pBspoIIB_Bs/ΔspoVG at T₁, T₃, and T₁₇. The cell membrane is visible as red fluorescence. Yellow and white arrows indicate the asymmetric septum and engulfed cells (prespores), respectively. (b–d) Relative mRNA expression of genes spoIIM, spoIIQ, and cotE determined in the ΔspoVG, RΔspoVG, and pBspoIIB_Bs/ΔspoVG strains compared with strain A16R (wild-type) at T₁₇ using RT-qPCR. Values represent means ± SDs of at least two experiments.

Figure 7

Schematic representation of regulatory pathways and their effectors in B. anthracis and B. subtilis based on the results of this study. (a) In B. subtilis, SpoVG is involved in spore formation at multiple stages (asymmetric division, engulfment, and cortex formation). Combined mutation of spoIIB and spoVG prevents spore formation at the engulfment stage. (b) In B. anthracis, the ΔspoVG mutant shows no asymmetric septum formation. SpoVG positively modulates spore formation through SpoIIE. B. subtilis SpoIIB (SpoIIB_Bs) partly restored spore formation in the B. anthracis ΔspoVG strain at the engulfment stage of sporulation.