Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis

Timothy M Hermanas; Sundharraman Subramanian; Charles E Dann, III; George C Stewart

doi:10.1128/JB.00135-21

. 2021 Aug 9;203(17):e00135-21. doi: 10.1128/JB.00135-21

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis

Timothy M Hermanas ^a,^b,^#, Sundharraman Subramanian ^c,^#, Charles E Dann III ^c,^✉, George C Stewart ^a,^b,^✉

Editor: Michael Y Galperin^d

PMCID: PMC8351633 PMID: 34096779

ABSTRACT

Bis-(3′-5′)-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore.

IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

KEYWORDS: Bacillus anthracis, anthrax toxin, c-di-GMP, exosporium, germination, spore, sporulation

INTRODUCTION

The bacterial second-messenger cyclic di-GMP (c-di-GMP) plays an important role in the control of bacterial motility, biofilm formation, and virulence gene expression (1, 2). In general, an increase in intracellular c-di-GMP levels leads to a sessile biofilm lifestyle, whereas low c-di-GMP levels favor a planktonic motile lifestyle. Cellular levels of c-di-GMP are regulated by synthesis of c-di-GMP from two molecules of GTP by diguanylate cyclases (DGCs) and hydrolysis of c-di-GMP into the linear dinucleotide 5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG) and/or GMP by c-di-GMP-specific phosphodiesterases (PDEs). The proteins responsible for DGC activity can be identified by the presence of GGDEF domains and PDEs by the presence of an EAL or HD-GYP domain (3). c-di-GMP modulates downstream processes by binding to a variety of effectors, including PilZ domain proteins, degenerate GGDEF/EAL domain proteins, transcription factors, and riboswitches (4). Although the c-di-GMP system has been better characterized with Gram-negative bacteria, recent reports now show that c-di-GMP mediated signaling exists in Gram-positive bacteria, including Bacillus amyloliquefaciens (5), Bacillus subtilis (6, 7), Bacillus thuringiensis (8 –10), Bacillus cereus (11), Clostridioides difficile (12 –15), Streptomyces spp. (16 –20), and Listeria monocytogenes (21 –23).

Among the spore-forming bacteria, c-di-GMP levels regulate motility, biofilm formation, toxin production, and intestinal colonization by C. difficile (12 –15). In Streptomyces spp., high c-di-GMP levels affect sporulation through inhibition of aerial-hypha formation and decreased production of antibiotics (16 –20). In the genus Bacillus, bioinformatic approaches have identified a range of putative c-di-GMP-related proteins encoded in Bacillus sp. genomes, ranging from 59 c-di-GMP-related proteins in Bacillus selenitireducens MLS10 to only two EAL or HD-GYP domain-containing proteins in Bacillus clausii KSM-K16 (https://www.ncbi.nlm.nih.gov/Complete_Genomes/c-di-GMP.html) (3). Levels of c-di-GMP regulate B. subtilis swarming motility and exopolysaccharide formation but not biofilm formation (24, 25), whereas control by c-di-GMP of motility and biofilm production was documented for B. amyloliquefaciens (5). Motility, biofilm formation, and toxin production were responsive to c-di-GMP levels in B. thuringiensis (9). The authors also noted modest opposing impacts on sporulation timing and spore yield associated with the cdgC (predicted to be an enzymatically inactive DGC) and cdgJ (predicted to be an inactive PDE) determinants. The DGC/PDE gene content and potential roles in the life cycle of the zoonotic pathogen Bacillus anthracis have not been described and are the subjects of this study.

RESULTS

In vivo characterization of individual c-di-GMP-metabolizing enzymes.

Bacillus anthracis Sterne has 10 predicted c-di-GMP metabolic enzymes, of which six proteins have both GGDEF and EAL domains (Fig. 1). One HD-GYP domain protein, a potential c-di-GMP phosphodiesterase, was also proposed from bioinformatics analysis. Three predicted nucleotidyl cyclase III domain proteins, Bas3907 (CdgC [9]), Bas5086 (CdgL [9], with a highly degenerate GGDEF domain with a subthreshold match to the GGDEF Pfam family), and Bas5323, are also present. The former two are likely mononucleotidyl cyclases, and the latter has a GdpP domain and is a likely c-di-AMP phosphodiesterase. These three determinants are not characterized further here.

The functional characterization of the B. subtilis c-di-GMP signaling pathway, combined with the presence of a clear biological consequence of high c-di-GMP levels (loss of motility), led to the development of mutant strains that can be used to monitor c-di-GMP metabolic enzymes from other microorganisms (6). The mutant strains contain an extra copy of dgrA to increase c-di-GMP receptor concentration and thus exhibit a strong motility phenotype. The mutant strain NPS236 has no c-di-GMP metabolic enzymes (i.e., it is motile) and can be used for screening heterologous DGCs. Active DGCs would be identified by a reduction in motility. The mutant strain NPS235, having no phosphodiesterase activity to limit the accumulation of c-di-GMP (i.e., being nonmotile), can be used for screening heterologous PDEs, via increases in motility. A chimeric c-di-GMP sensing riboswitch was positioned upstream of the coding sequence of green fluorescent protein (GFP) to sense c-di-GMP levels directly in strains NPS400 and NPS401 (6). This is an “off” switch, so high c-di-GMP levels result in low GFP signal. The B. anthracis genes listed in Fig. 1 were cloned into the plasmid pXG101 downstream of the IPTG-inducible P_hyspank promoter and introduced into the B. subtilis reporter strains as single-copy chromosomal insertions at the thrC locus. All coding sequences were constructed as a translational fusion to the B. subtilis dgcP leader to ensure proper transcription and translation initiation in the heterologous host (6).

Five of the putative DGCs tested were capable of inhibiting swarming motility, indicative of active DGCs possessing the ability to produce c-di-GMP (CdgA, CdgB, CdgD, CdgG, and CdgI) (Fig. 2) when expressed in strain NPS236 derivatives. Reduction of riboswitch-regulated GFP expression in the strain NPS400 background confirmed elevation of c-di-GMP with expression of these genes. Expression in the strain NPS235 background revealed three active phosphodiesterases (CdgD, CdgE, and CdgH) capable of reducing c-di-GMP levels, thus increasing swarming motility and reducing c-di-GMP levels and thus elevating GFP production in strain NPS401 (Fig. 3). CdgD is both an active DGC and PDE. Frequently, one activity dominates, such that the second domain of putative bi-functional enzymes appears inactive in any one experiment, although examples of dually active enzymes have been described in the literature (2, 9, 26, 27). CdgB is an active diguanylate cyclase without any predicted sensor domain. The enzymes identified as putative DGCs or PDEs were classified as active or inactive following heterologous expression in B. subtilis. The enzymes for which DGC or PDE activities were not demonstrated could potentially be active under unique environmental conditions or in the presence of host-specific components not tested here.

FIG 2 — Active DGCs from *B. anthracis* Sterne. Comparison of normalized GFP fluorescence (single-copy expression of indicated genes in NPS236) and normalized swarm radius of *B. subtilis* NPS236 derivative strains expressing putative *B. anthracis* diguanylate cyclases based on the presence of GGDEF domains. Active proteins resulting in reduced motility or c-di-GMP-mediated reduction of GFP expression are indicated with green stars. **, P < 0.01; ****, P < 0.0001.

FIG 3 — Active PDEs from *B. anthracis* Sterne. Comparison of normalized swarm radius of *B. subtilis* NPS235 strains expressing putative *B. anthracis* phosphodiesterases, based on the presence of EAL or HD-GYP domains. Levels of GFP production were measured by FACS in strains of NPS401 bearing the indicated genes recombined into the *thrC* locus. Active proteins lowering c-di-GMP levels, resulting in increased motility and reduced GFP expression, are indicated with green stars. **, P < 0.01; ****, P < 0.0001.

Expression of the GGDEF/EAL determinants in B. anthracis Sterne.

All of the putative c-di-GMP genes listed in Fig. 1 were individually cloned into the pDG4100 shuttle plasmid to create C-terminus fusions to mCherry, with expression regulated using the native promoter and ribosome binding site for each gene. The resulting plasmids were individually electroporated into B. anthracis Sterne. The transformed strains (MUS8200-8209 and control strains bearing the pDG4100 vector plasmid [MUS8210], the BclA exosporium nap glycoprotein N-terminal domain-mCherry fusion plasmid [MUS8181], and the plasmid expressing unfused mCherry from the bclA promoter [MUS8159]) were cultured in nutrient broth, and expression of the fluorescent fusion protein was monitored throughout the growth and sporulation phases by fluorescence microscopy and Western immunoblot analysis using anti-mCherry antibodies. The cdgE, cdgF, cdgH, and cdgI fusion determinants were not sufficiently expressed when introduced on plasmids in B. anthracis Sterne to produce detectable fluorescent protein throughout their in vitro life cycle (see Fig. S1 in the supplemental material). The cloned genes were sequenced to ensure that no mutations were introduced during the cloning or electrotransformation processes, and no sequence changes were found (data not shown). The expression profiles for the other putative c-di-GMP genes are shown in Fig. 4. No signal on the Western immunoblots was observed with the Sterne strain bearing the pDG4100 vector plasmid, and Sterne bearing pGS4709 (mCherry transcribed from the bclA promoter) produced the mCherry unfused protein only during the sporulation phase time points (Fig. S1). Four of the six examined determinants were expressed at both the mid-logarithmic growth phase (optical density at 600 nm [OD₆₀₀] = 0.6) and during sporulation, whereas two (cdgA and cdgB) were expressed only during sporulation time points, with the latter being evident beginning at T₇ (7 h into the sporulation phase) and the former appearing at T₃ (although not intact, as evidenced by its appearing as a smaller and more heterogeneously sized band) and more stably present at T₄ and later time points. Smaller species, likely degradation products, were evident at the later sporulation time points (CdgA, CdgB, and CdgM), at early sporulation and mid-exponential phase (CdgJ), or throughout sporulation (CdgD and CdgG). Interestingly, the CdgD, CdgG, CdgM, and to a lesser extent CdgA fusion proteins were incorporated into high-molecular-weight complexes that resisted disruption by boiling in SDS in the presence of reducing agent. Formation of this type of high-molecular-weight complex is a characteristic of certain B. anthracis spore structural proteins (28, 29).

FIG 4 — Anti-mCherry Western immunoblot analysis of *B. anthracis* Sterne mCherry fusion lysates. Lysates from cells collected at different time points during *in vitro* culture in nutrient broth were subjected to SDS-PAGE, and Western immunoblots were probed with rabbit polyclonal antiserum against mCherry. Lane 1, lysate at an OD₆₀₀ of 0.6 (mid-exponential phase); lanes 2 to 13, sporulation time points T₁ to T₁₁. The migration position of the fusion protein is indicated with arrows. Molecular weights of the fusion proteins are as follows: CdgA-mCherry, 64,800; CdgB-mCherry, 78,800; CdgD-mCherry, 133,000; CdgG-mCherry, 132,000; CdgJ-mCherry, 76,000; and CdgM-mCherry, 72,800.

Detection of mCherry fusion proteins on or within B. anthracis spores.

Purified spores from the fusion protein-containing strains were examined by fluorescence microscopy. Spores from Sterne harboring only the empty expression vector pDG4100 lacked identifiable fluorescence (Fig. S2). Spores from he B. anthracis Sterne pGS4709, which expresses unfused mCherry expressed from the bclA promoter (high levels of expression during sporulation), also lacked detectable fluorescence on the mature spores. The presence of the unfused mCherry protein within sporulating cells and its absence on spores suggests that production of mCherry during sporulation is not sufficient for protein incorporation into, or passive adherence to, mature spores. The CdgA, CdgB, CdgD, CdgG, CdgJ, and CdgM fusion proteins showed evidence of incorporation into spores with varying intensity (Fig. S2).

To determine if the fusion proteins were incorporated into the exosporium layer of the spore, the plasmids were introduced into the B. anthracis Sterne cotE mutant. Loss of cotE results in spores that form an exosporium that fails to attach to the spore coat, resulting in its sloughing off when mother cell lysis occurs (30). If spores lose fluorescence in the B. anthracis ΔcotE background, it suggests the fusion proteins are located at the exosporium layer of the spore. The CdgA, CdgB, CdgD, CdgG, CdgJ, and CdgM fusion proteins retained fluorescence in cotE-negative spores, suggesting they are not located in the exosporium layer (Fig. 5). Fluorescence microscopy of spores from the strain expressing the targeting domain of the exosporium nap layer protein BclA fused to mCherry showed that it was abundantly expressed on wild-type spores, but within the cotE background, the spores lacked fluorescence but fluorescence was evident on the exosporium strips separated from the spores.

FIG 5 — Micrographs of *B. anthracis* Sterne and *B. anthracis* Sterne *cotE* spores expressing mCherry fusion proteins. Spores were diluted to 100 μg/μl and 2 μl of spores were diluted in 10 μl of PBS, spotted onto glass microscope slides, and allowed to settle. Spores were visualized by bright-field (left) and fluorescence (inverted image, center) microscopy; the merged image is on the right. Inverted and merged images were made using ImageJ.

B. anthracis biofilm production.

There are reports of B. anthracis forming biofilms under static and flow cell conditions (31, 32). Despite these studies, whether B. anthracis can form biofilms in the natural environment is controversial (33). We attempted to produce static biofilms using polystyrene plates and examined B. anthracis Sterne at 12, 24, 48, and 72 h in brain heart infusion broth (BHI), Luria broth, tryptic soy broth, nutrient broth, BHI plus 0.5% glucose (BGGM), 2× YT broth, and maltose broth. None of the conditions gave rise to biofilm formation by B. anthracis (Fig. S3A), nor was there evidence of a pellicle biofilm at the air-liquid interface. Despite the poor biofilm-forming properties of the wild-type Sterne strain, we tested for biofilm formation by the c-di-GMP system deletion mutants. The rationale was that loss of phosphodiesterase activity might result in elevation of c-di-GMP levels and in a more robust biofilm phenotype. Deletion mutations involving the cdgD and cdgE determinants of B. thuringiensis resulted in increased biofilm production, providing support for this approach (9). The results of our experiments are presented in Fig. S3B. None of the mutant strains exhibited a biofilm-forming phenotype. The lack of a strong biofilm phenotype of the Sterne strain and the results obtained herein may suggest that there is not a role for c-di-GMP for biofilm formation in B. anthracis. However, deliberate overexpression of the DGCs would be required to confirm this.

Anthrax toxin production.

B. anthracis produces a tripartite toxin composed of protective antigen (PA), lethal factor, and edema factor essential for virulence in mammalian hosts (34). Because elevated levels of c-di-GMP are known to suppress toxin production in several bacterial species (1 –4), the ten deletion mutants were examined for effects on secreted PA levels using in vitro assays. All strains were analyzed by PCR to confirm the presence of the pag determinant encoded on the pXO1 plasmid and confirm the absence of pag in B. anthracis ΔSterne, which lacks the pXO1 plasmid. PA is secreted with a molecular weight of 83 kDa (35). Levels of secreted PA were assessed by Coomassie blue straining and Western immunoblotting with rabbit anti-PA polyclonal antiserum (Fig. 6). B. anthracis Sterne and all ten deletion mutants produced PA in similar amounts. The amounts relative to B. anthracis Sterne are shown in Fig. 6C. None of the deletion mutations had a detectable impact on PA, hence anthrax toxin, expression in vitro.

FIG 6 — Toxin production in GGDEF/EAL/HD-GYP mutants. Trichloroacetic acid-precipitated supernatants from broth cultures were analyzed by Coomassie brilliant blue R-250 staining (A) and Western immunoblotting using a rabbit anti-PA polyclonal antibody (B). Lanes: (+), *B. anthracis* Sterne; (−), *B. anthracis* ΔSterne; 1, Δ*cdgH*; 2, Δ*cdgF*; 3, Δ*cdgM*; 4, Δ*cdgG*; 5, Δ*cdgE*; 6, Δ*cdgJ*; 7, Δ*cdgB*; 8, Δ*cdgD;* 9, Δ*cdgI*; 10, Δ*cdgA*. rPA, recombinant PA (5 μg). (C) The densities of the bands obtained using anti-PA antibody were quantified using NIH ImageJ. The pixel density was normalized by dividing the Sterne pixel density and multiplying by 100 to give the percent. Error bars represent the standard deviations from three independent experiments.

BclA incorporation into spores.

BclA makes up the outermost layer of the B. anthracis spore and is the structural component of the exosporium hair-like nap (36). BclA is attached to the basal layer though an N terminus targeting domain (NTD) (37). Fusing the NTD to dsRed and examining insertional transposon mutants that exhibited fluorescence during the early phase of sporulation but lost fluorescence after cell lysis allowed the identification of putative genes whose products impact BclA attachment to the basal layer (38). Spreng et al. identified ten Tn916 insertion mutants exhibiting this phenotype (38). One transposon insertion site was localized to the gene encoding the putative GGDEF/EAL domain-containing protein CdgE. Therefore, incorporation of BclA-NTD-mCherry into spores produced by all ten deletion mutants was examined using flow cytometry and immunofluorescence microscopy. Spores of B. anthracis Sterne exhibited robust staining (Fig. S4). None of the deletion mutants exhibited altered expression of BclA on the spore surface. Thus, these genes likely play no role in BclA assembly onto the exosporium surface, and the results suggest that the effect observed with the transposon study may not have been due to loss of cdgE but was perhaps due to an unanticipated effect of the transposon insertion at this site on the B. anthracis chromosome.

Sporulation efficiency.

Fagerlund et al. (9) screened all B. thuringiensis 407 GGDEF/EAL/HD-GYP determinants and found that a cdgJ deletion mutant had a delayed timing of sporulation and that presumed overexpression of cdgJ resulted in an earlier accumulation of spores relative to the wild-type strain. Conversely, deletion of cdgC resulted in an earlier sporulation. The B. thuringiensis cdgC and cdgJ determinants are predicted to encode enzymatically inactive DGC and PDE proteins, respectively, and in the cases of gene inactivation or overexpression, the difference in number of spores obtained relative to the wild-type strain did not reach statistical significance. To determine if the c-di-GMP system of B. anthracis impacted sporulation, we examined sporulation efficiencies in our collection of GGDEF/EAL/HD-GYP mutant strains. The sporulation competence of B. anthracis Sterne was compared to that of the deletion mutants to identify mutants that may be affected in the sporulation process at an early time point (12 h) and after 1, 2, and 3 days of incubation. The results are shown in Fig. 7. No significant differences were noted when the results were analyzed by analysis of variance (ANOVA), with P values of 0.867, 0.989, 0.219, and 0.479 for results at 12, 24, 48, and 72 h, respectively.

FIG 7 — Sporulation efficiencies. Overnight cultures of *B. anthracis* Sterne and deletion mutants were inoculated 1:500 into 50 ml of prewarmed nutrient broth, and spore formation was assessed at 12, 24, 48, and 72 h of incubation at 37°C. Viable counts were determined on culture samples with and without heat treatment (65°C for 30 min to kill vegetative cells). Spore titers were determined by dividing the number of heat-resistant CFU by the number of untreated CFU. Error bars denote the standard deviations from three independent experiments.

The effects of the c-di-GMP system determinants on spore germination efficiencies.

The spore is the infectious form of B. anthracis. Infection of a host results in uptake of the spores by phagocytic cells followed by germination of the spores and replication of the vegetative bacteria accompanied by toxin production. Timing of germination is important in B. anthracis in the initial stages of the infectious process, with early germination resulting in higher intracellular killing of the emerging bacteria (39). Therefore, we tested the germination properties of the c-di-GMP-associated gene deletion mutants. The initial stages of germination in response to the germinants l-alanine and inosine were measured in a spectrophotometric assay, and the results are shown in Fig. 8. Six of the deletion mutants, the ΔcdgD, ΔcdgG, ΔcdgH, ΔcdgI, ΔcdgJ, and ΔcdgM mutants, exhibited wild-type germination kinetics (Fig. 8A). Four of the mutants reproducibly exhibited altered germination kinetics. Spores from the ΔcdgF strain germinated more rapidly than spores from the Sterne parent strain (Fig. 8B). Introduction of the cdgF allele on a pHP13-based shuttle plasmid into this mutant only partially restored the germination kinetics. Paradoxically, addition of the complementation plasmid into the Sterne strain resulted in an increase, rather than the expected decrease, in the germination kinetics to the level observed with spores from the ΔcdgF mutant bearing the complementation plasmid.

The ΔcdgE mutant spores exhibited a decreased germination kinetics compared to the parent Sterne strain (Fig. 8C). Introduction of the plasmid-borne wild-type allele into the ΔcdgE resulted in a slight, but reproducible, complementation of the germination phenotype. The addition of this plasmid into the wild-type Sterne strain also resulted in a modest increase in the germination kinetics.

The ΔcdgB mutant spores exhibited a modest increase in germination kinetics relative to the Sterne parent spores (Fig. 8D). Addition of the plasmid bearing the wild-type allele resulted in markedly faster germination of both Sterne and the ΔcdgB mutant host strains.

The ΔcdgA mutant spores exhibited a decrease in germination kinetics relative to the Sterne parent spores (Fig. 8E). Addition of the plasmid bearing the wild-type cdgA allele resulted in reduced germination kinetics to comparable levels in both the wild-type and mutant host strains.

DISCUSSION

c-di-GMP is known to play an important role in several bacterial phenotypes, such as biofilm formation, motility, and toxin production. The intracellular concentration of c-di-GMP acts to turn these systems on or off in in a wide variety of Gram-negative and Gram-positive bacteria. In Bacillus species, an increase in c-di-GMP concentration has been shown to shut down motility and toxin production (9, 10, 24).

B. anthracis possesses ten shared genes coding for putative proteins with c-di-GMP synthesis and degradation activities, which were the focus of this study. Thirteen genes (cdgA to cdgM) were identified in the B. cereus group genomes, of which eleven are present in the B. thuringiensis 407 genome (9). Twelve were identified in the B. thuringiensis BMB171 genome (10). The cdgK determinant is not present in the B. anthracis Sterne genome, and its presence is variable among members of the B. cereus group (9). The cdgC and cdgL genes are present in the Sterne genome, but evidence for their having a role in c-di-GMP metabolism is currently lacking. Using a B. subtilis model system for evaluating DGC and PDE activity, we identified four functional DGCs, three functional PDEs, and one functional HD-GYP protein. One protein, CdgD, possessed both active GGDEF and EAL domains. The B. cereus CdgF protein (from strain ATCC 14579) was shown to have this dual activity in an in vitro analysis (9) but was characterized as inactive for both activities in this study. The B. anthracis Sterne and the B. thuringiensis 407 proteins are 95% identical (537/567 residues) and 97% similar (553/567 residues). In the in vitro analysis, the DGC activity was maximal when the associated flavin cofactor was oxidized, and when it was in a reduced state, DGC activity decreased and EAL-mediated PDE activity increased (9). The redox environment of the B. subtilis host cells may have affected the enzymatic activities of the CdgF protein.

B. anthracis was found to be a poor biofilm producer, using a variety of media. If the c-di-GMP regulatory network impacted biofilm production, then certain deletions of the genes associated with this pathway would have been predicted to increase biofilm production. However, screening of all ten mutants revealed no increase in biofilm levels. This is not necessarily an unexpected finding. Whereas B. subtilis and B. thuringiensis are like B. anthracis in that they are soil spore-forming organisms, B. anthracis differs in that it does not replicate extensively in soil as a saprophyte. Controlled spore germination and replication studies in soil environments with B. anthracis consistently demonstrate that although spore germination can occur under certain environmental conditions, the numbers of spores and vegetative cells do not increase substantially (40 –42). This suggests that replication of the emerging cells and/or subsequent sporulation are not efficient in these soil environments. Studies with B. subtilis have shown that biofilm formation is an important physiological trait for bacterial replication in soil. Studies have shown that biofilm formation is important for stable colonization of the plant root, an association that is mutually beneficial for the bacterium and the plant (reviewed in reference 43). Biofilm formation in soil bacteria impacts acquisition of water and competition with other soil microbes (44, 45). Because B. anthracis does not actively replicate in soil, it may not have been subject to the evolutionary pressures to develop or maintain a biofilm life style. The lack of significant replication in soil explains the slow evolutionary clock exhibited by B. anthracis strains in that the organism spends the majority of its time as a metabolically inert spore in soil (46). When the B. anthracis organisms do replicate, it is intracellularly in phagocytic cells during the initial stages of infection or within the bloodstream or lymph of infected animals. Forming biofilms provides no apparent advantage in these environments, negating selective pressure to evolve into biofilm producers. The cdgF determinant of Bacillus thuringiensis plays an essential role in biofilm formation in B. thuringiensis (9). It was shown to have the strongest impact on c-di-GMP levels, and transcription of this gene was higher than that of the other cdg determinants. It is interesting that our results and those of Bergman et al. (47) indicate that cdgF is poorly expressed by the poorly biofilm-producing B. anthracis.

When the ten deletion mutants were screened for toxin production, no change was seen in the mutants when they were compared to the parent strain. The production of anthrax toxin is regulated in binary fashion by the anthrax toxin activator A (AtxA) protein, which responds to increases in temperature, CO₂ levels, and bicarbonate levels during host infection (48). In other bacterial pathogens, the c-di-GMP regulatory network oppositely regulates toxin production and biofilm formation. Since there is no biofilm-versus-planktonic-cell dichotomy with this pathogen, a mechanism for c-di-GMP controlling toxin production would not be needed.

Sporulation efficiency was also assayed in each of our ten deletion mutants, and no significant change in phenotype was observed. This is a slightly different observation than that of a study performed with B. thuringiensis where two cdg genes predicted to encode enzymatically inactive proteins (cdgC and cdgJ) had an impact on sporulation (9). A cdgJ deletion mutant exhibited a delayed timing of sporulation, and introduction of cdgJ on a multicopy plasmid in an otherwise wild-type host resulted in an earlier accumulation of spores relative to the wild-type plasmid-free strain (although these differences were not statistically significant). Conversely, deletion of cdgC, carrying a degenerate GGDEF motif, resulted in an earlier onset of sporulation, and the relative number of spores after 20 h was more than twice that of the wild-type control, although the spore number difference at 24 h was not statistically significant (9). Sporulation is a process which in B. subtilis has been shown to be linked to biofilm formation, with respect to both the early sporulation phase transcriptional regulator Spo0A taking part in both processes and spores constituting natural parts of a developing B. subtilis biofilm (49). The lack of a strong biofilm phenotype with B. anthracis may negate the need for a role for c-di-GMP signaling in sporulation. The timing and commitment of sporulation in B. anthracis also differ from those in other saprophyte sporeformers, because sporulation is triggered by an abrupt change from a nutrient-rich milieu of the bloodstream to deposition into a much less rich soil surface environment, rather than the more gradual nutrient shifts that would be expected for bacteria growing in soil environments.

Control of germination and its timing is critical for B. anthracis during early events following introduction of spores into an infected host. For example, spores contain the enzyme Alr, which converts l-alanine to d-alanine to prevent premature germination in macrophages (50). Loss of Alr results in germination at lower l-alanine germinant concentrations. Alr-deficient strains exhibited reduced survival in in vitro macrophage infection assays and impaired survival in in vivo mouse infections (51). Testing the early phase of germination in this study identified four genes which resulted in altered germination kinetic phenotypes when deleted or introduced on a multicopy plasmid. The cdgE gene, which encodes a protein with an active EAL (phosphodiesterase) domain, is transcribed in a biphasic pattern with expression during exponential-phase growth and during sporulation (47) (Fig. 9). Deletion of the gene, which might elevate c-di-GMP levels, resulted in decreased germination kinetics. Introduction of the complementing plasmid, possibly reducing c-di-GMP levels, increased germination kinetics. This result suggests that higher levels of c-di-GMP in the sporulating cells leads to a more efficient response to germinants by the mature spores. However, two genes encoding active DGCs, cdgA and cdgB, were found to impact germination kinetics. Mutants with deletions of these genes gave opposite results. The cdgA mutant exhibited reduced germination kinetics, whereas the cdgB mutant germinated more quickly. The presence of plasmid-borne cdgA further reduced germination kinetics, and plasmid-borne cdgB further increased germination kinetics. Thus, the cdgA and cdgB genes have opposite effects. The CdgA and CdgB proteins are both present during the sporulation process, but the transcription profiles for the genes are distinct. The cdgA gene is transcribed at low levels throughout the in vitro growth and sporulation phases, and the cdgB gene is highly expressed only late in the sporulation phase (47) (Fig. 9).

FIG 9 — Transcription of the *B. anthracis* c-di-GMP determinants (microarray data from reference 47). Onset of sporulation in this study began at approximately 240 min. The expression of the *sigK* determinant, which encodes the sigma K factor responsible for transcription of late sporulation genes in the mother cell, is shown as a sporulation time point reference. (Left) Genes that are not appreciably expressed during sporulation; (right) genes that are appreciably expressed during sporulation.

Regulation of spore responses to germinants is not fully understood. Membrane-associated germinant receptors are expressed during the sporulation phase, but it is unknown how these are regulated beyond transcription by sporulation-phase sigma factors. Involvement by c-di-GMP levels would likely involve not only levels of the c-di-GMP metabolic proteins but also the timing of their expression and where the genes are expressed (in the forespore or mother cell compartment).

The ΔcdgF mutant strain exhibited a large increase in germination rate relative to the parent Sterne strain, but the presence of the wild-type allele on a plasmid also produced modest increases in germination rate in the Sterne parent strain. The CdgF protein possesses inactive GGDEF and EAL domains based on the B. subtilis assay system but a largely identical homolog from B. cereus was shown to have DGC and PDE activities in vitro (9). Expression levels of the cdgF gene are very low when the bacteria transition to sporulation phase (47) (Fig. 9). It remains, therefore, unclear if CdgF has an impact on c-di-GMP levels in the context of B. anthracis cells during the process of spore formation.

Six of the Cdg proteins were found to localize to spores in a CotE-independent manner. The presence of c-di-GMP pathway proteins in spores is not surprising given that transcription of a subset of these determinants has been shown to occur during sporulation (47) (Fig. 9). Notably, transcription of cdgB was shown by Bergman et al. (47) to be induced in late sporulation phase, cdgA showed a modest increase in early sporulation phase, and cdgE was biphasic in its expression pattern with log-phase and sporulation-phase peaks in expression. Notably, these determinants exhibited effects on germination rates in this study. The different timing of expression of these genes may result in localization of these proteins in different compartments of the developing spore. In this study, the inactive PDE protein CdgJ and the active HD-GYP protein were expressed throughout the in vitro life cycle and were incorporated into spores. CdgA, CdgB, CdgD, and CdgG have active GGDEF domains and were expressed during the sporulation phase. CdgD, with a weakly active EAL domain when tested in the heterologous B. subtilis swarming assay, was also found to be spore-associated. This suggests that c-di-GMP may play a role in the process of germination during the initial stages of B. anthracis infection or in the initial host-pathogen interactions following the intracellular germination of the spores in the course of an infection. If the GGDEF domains are active in spores or if c-di-GMP is produced and released after spores germinate within these cells, it could trigger an immune response through interaction with STING, which results in a type I interferon response (52).

The activation of a host innate immune receptor could, under certain circumstances, be beneficial to the pathogen. Listeria monocytogenes has multidrug efflux pumps that secrete c-di-AMP, which then activates STING (53). The decreased protective immunity was largely dependent on the type I interferon receptor, suggesting that L. monocytogenes activation of STING downregulates cell-mediated immunity by induction of type I interferon. A potential activation of STING via c-di-GMP may be part of a survival strategy of B. anthracis following spore germination in macrophages or dendritic cells during the initial stages of the infection.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Bacterial strains and plasmids are listed in Table S1 in the supplemental material. The Sterne strain of B. anthracis is the pXO2-negative vaccine strain. Escherichia coli strains were cultivated using Luria broth (LB). Bacillus anthracis and Staphylococcus aureus were grown using brain heart infusion agar and broth (Difco). Agar plates were made by the addition of agar at a concentration of 1.5% (wt/vol). Nutrient broth and agar (Oxoid) were used for sporulation. B. subtilis strains were cultured in LB medium at 37°C with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) in cases where the P_hyspank promoter was induced. For biofilm studies, 2× YT broth (per liter: 16 g tryptone 10 g yeast extract, 5 g NaCl) and maltose broth (per liter: 10 g tryptone, 1 g yeast extract, 5 g NaCl, 10 g maltose) were also used. The following antibiotics, where needed, were added (final concentration): ampicillin (100 μg/ml), chloramphenicol (10 μg/ml), kanamycin (25 μg/ml), and spectinomycin (100 μg/ml).

Identification of the c-di-GMP determinants of B. anthracis.

Sequences of the B. anthracis Sterne proteins containing significant matches to the Pfam GGDEF (PF00990), EAL (PF00563), or HD-GYP (PF01966) domain protein families were downloaded from the Pfam server (http://pfam.xfam.org/). These sequences were used to search the B. anthracis Sterne genome (NCBI accession no. AE017225.1). Nucleotide and protein accession numbers are provided in Table S2.

DNA purification.

The Wizard SV miniprep kit (Promega) was used to isolate plasmid DNA. For B. anthracis the pellets from 5-ml cultures were frozen at −80°C overnight and thawed at 37°C prior to DNA extraction. Genomic DNA was isolated using the Wizard Genomic DNA purification kit (Promega). For B. anthracis, the cell pellets were frozen at −80°C overnight and thawed at 37°C prior to DNA extraction.

Construction of B. subtilis strains expressing the B. anthracis putative c-di-GMP determinants.

To generate inducible translational fusion constructs for genes encoding putative diguanylate cyclases and c-di-GMP phosphodiesterases from B. anthracis Sterne, the gene open reading frames were PCR amplified from B. anthracis Sterne genomic DNA (primers are listed in Table S3). The predicted DGCs and PDEs were cloned into the plasmid pXG101, which has an IPTG-inducible P_hyspank promoter along with B. subtilis dgcP leader peptide (−60 to +3 relative to the translation start site) (6). The cloned genes in the plasmid are flanked by segments of thrC gene for homologous recombination. Constructs were then introduced by transformation into the B. subtilis strain DS2569 and the transformants were confirmed by selection on minimal medium plates without threonine (6). Using SPP1 phage mediated transduction, genes were then introduced into the respective target strains (24). The predicted diguanylate cyclase proteins were introduced into the strain NPS236 for swarm assays and NPS401 for fluorescence-activated cell sorting (FACS) analysis. The predicted phosphodiesterase proteins were introduced into strain NPS235 for swarm assays and NPS400 for FACS analysis.

B. subtilis swarm expansion and c-di-GMP-responsive riboswitch-GFP reporter assays.

Motility swarm expansion assays and FACS analyses of riboswitch-GFP reporters were as previously described (6, 24). Data were subjected to one-way analysis of variance (ANOVA) using Dunnett's multiple-comparison test with at least three measurements for each data point using Prism 6 software. Fluorescence data are normalized to same-day control strains in each experimental set.

Electroporation of B. anthracis.

To prepare B. anthracis electrocompetent cells, bacteria from an overnight BHI agar plate were inoculated into 50 ml of prewarmed BHI plus 0.5% glycerol and incubated at 37°C with shaking until the OD₆₀₀ reached ∼0.6 to 0.8. The culture was then passed through a disposable analytical test filter funnel apparatus with a pore size of 0.45 μm (Thermo Scientific). The filtered cells were then washed twice with 20 ml of ice-cold electroporation buffer (1 mM HEPES, 10% glycerol [pH 7.0]). The filter was then placed in a 50-ml polypropylene conical tube, and the cells were washed off the filter by vortex mixing with 3 ml electroporation buffer. Cells were aliquoted into amounts of 400 μl and used immediately or stored at −80°C. For electrotransformation, 10 μl (∼1 microgram) of plasmid DNA was mixed with 200 μl of electrocompetent B. anthracis cells. All plasmids used for electroporation were passed through E. coli GM48 to produce DNA lacking the Dam methylation pattern. The samples were incubated for 10 min on ice, transferred to a chilled 1-mm electroporation cuvette (Midwest Scientific), and shocked with a pulse of 2.25 kV, 25 μF, and 100 Ω with a Bio-Rad Gene Pulser apparatus. The bacteria were transferred to a 15-ml polypropylene conical tube with 2.5 ml of BGGM (BHI with 10% glycerol, 0.4% glucose, and 10 mM MgCl₂) and incubated at 37°C for 60 min (or 30°C for 90 min for temperature-sensitive plasmids). Samples were then plated onto BHI agar plates with appropriate antibiotics and were incubated at 37°C or 30°C overnight.

Construction of the gene fusions to mCherry.

The mCherry fusions were generated by PCR amplifying the B. anthracis genes using primers with a 5′ SacI restriction site upstream of the native promoter element and a 3′ NheI site immediately prior to the termination codon of the open reading frame. SacI- and NheI-digested DNA fragments were then cloned into identically digested pDG4100 and transformed into E. coli strain DH5α. The plasmid pDG4100 is a derivative of the pMK4 shuttle plasmid (54) into which has been inserted the mCherry open reading frame (ORF) with an NheI site immediately adjacent to the 5′ end of the ORF and a SacI site upstream of the NheI site. Clones with plasmids of the correct size and restriction endonuclease pattern were identified, and their insert sequences were verified by nucleotide sequence analysis. The plasmids were then transformed into E. coli GM48, and plasmid DNA was isolated and electroporated into B. anthracis Sterne.

Generation of B. anthracis deletion mutants.

The gene deletion mutants were generated by PCR amplifying and cloning 1-kb sequences upstream and downstream of the GGDEF/EAL/HD-GYP open reading frame of interest. The upstream and downstream fragments were fused by splicing overlap exchange PCR. The resulting 2-kb fragment was cloned into SalI-digested and alkaline phosphatase-treated pGS4294. The allele exchange vector, pGS4294, is a 6.4-kb plasmid composed of pUC18 (lacking the BamHI and SmaI sites) joined with pE194 at their unique PstI sites. The pE194 replicon is temperature sensitive in Bacillus hosts. The plasmid confers resistance to ampicillin (in E. coli) and erythromycin (in B. anthracis). A spectinomycin resistance cassette was inserted into the BamHI site at the position of the deleted gene. Sequence-verified plasmids were isolated from E. coli GM48, electroporated into B. anthracis Sterne, and incubated at 30°C. Colonies exhibiting spectinomycin resistance were inoculated onto spectinomycin and erythromycin (the pGS4294 vector-encoded resistance) plates to ensure that no spontaneous spectinomycin-resistant colonies arose and that cells from the colony harbored the allele replacement plasmid. Following confirmation of both Spec^r and Ery^r of the transformants, the resulting B. anthracis clones were inoculated into 10 ml of BHI broth containing spectinomycin and grown overnight at 42°C with shaking. Thirty microliters of the culture was then plated on a BHI agar-spectinomycin plate and streaked for isolation of single colonies, which were grown overnight at 37°C. Larger colonies were selected with this semiselective incubation temperature. This process of growing an overnight liquid culture at 42°C and plating was repeated until PCR analysis of DNA from the clone using primers flanking the gene to be deleted gave the DNA fragment size corresponding to the deletion allele. Sequence analysis of the PCR fragment confirmed the deletion.

Construction of complementation plasmids.

Complementation plasmids were generated for the cdgA, cdgB, cdgE, and cdgF open reading frames by amplifying the ORF with its native promoter using primers that added SalI sites to the ends. The resulting amplicons were then cloned into the E. coli-Bacillus shuttle plasmid pMK4 and sequence verified.

Western blot analysis.

For Western blot analysis, a volume of cells normalized to an OD₆₀₀ of 1 was aliquoted and frozen at −80°C. Cells were thawed on ice and centrifuged at 15,000 rpm for 10 min to pellet the cells. The resulting pellets were resuspended in 250 μl urea–SDS-PAGE buffer (6 M urea, 62.5 mM Tris-HCl [pH 6.8], 2.5% SDS, 0.002% bromophenol blue, 0.71 M β-mercaptoethanol, 10% glycerol) and boiled for 10 min. Fifteen microliters was loaded onto a 15-well Mini-Protean TGX gel (Bio-Rad) and electrophoresed at 190 V in SDS buffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS [pH 8.3]). The proteins were electrotransferred to Immobilon membranes (Millipore Sigma) for 1 h on ice at 250 mA in 25 mM Tris-HCl, 192 mM glycine, 10% methanol. Membranes were blocked using SuperBlock (Thermo Fisher) and stained with a 1:20,000 dilution of anti-mCherry rabbit polyclonal antibody (Invitrogen) overnight at 4°C. The membranes were then washed with 1% Tween 20 in phosphate-buffered saline (PBS). Secondary staining was done with 1:20,000 anti-rabbit IgG–horseradish peroxidase (HRP) conjugate (Invitrogen) at room temperature for 3 h, and the membrane was washed three times with 1% Tween 20 in PBS for 15 min. Chemiluminescence was detected using the Pierce ECL Western blotting substrate (Thermo Fisher) and blue-sensitive autoradiography film (Midwest Scientific).

Production of spores.

Cells from a BHI broth culture were swab inoculated with the B. anthracis strain onto the surface of 150- by 15-mm Oxoid nutrient agar plates with the appropriate antibiotics. The cultures were incubated at 30°C for 5 days. The surface layer of bacterial growth was harvested with a sterile cotton swab, and the spores were dispersed in PBS. The spores were harvested by centrifugation at 15,000 rpm, and the upper, less dense, pellet layer containing lysed cell debris was removed by flushing the pellet using a pipettor, followed by aspiration of the supernatant, which was then discarded. The spore pellet was resuspended in PBS, and the pelleting-washing process was repeated until there was no evidence of vegetative cells or cell debris present (verified by phase-contrast microscopy). Dried spore pellets were weighed to determine weight of spores, by volume, and the purified spore pellets were adjusted to a concentration of 100 μg/μl using PBS and stored at room temperature.

Anti-BclA immunofluorescence and flow cytometry.

Ten micrograms of spores was fixed in 4% paraformaldehyde in PBS for 60 min and washed three times with PBS. The spores were pelleted by centrifugation, resuspended in SuperBlock, and incubated for 1 h at room temperature. Following blocking, the spores were centrifuged and resuspended in 1 ml SuperBlock with a 1:250 dilution of anti-BclA rabbit polyclonal antiserum and incubated for 2 h at room temperature (55). The spores were washed three times with 1 ml PBS, resuspended in 1 ml of SuperBlock with Alexa Fluor 568, and incubated at room temperature for 2 h in the dark. The spores were then washed three times with 1 ml PBS, resuspended in 200 μl PBS, and analyzed by microscopy using a Nikon E600 epifluorescence microscope with the mCherry filter set. The bright-field and fluorescence images were merged using the NIH ImageJ software program (https://imagej.nih.gov/ij/download.html). For flow cytometry, the samples were analyzed using a FACScan flow cytometer (Beckton Dickinson), and data were analyzed using Cell Quest analysis software (Beckton Dickinson) at the University of Missouri Cell and Immunobiology Core laboratory.

Sporulation assay.

Cells of B. anthracis were inoculated into BHI broth and incubated at 37°C for 12 h with shaking. The cultures were diluted 1:500 into 50 ml prewarmed nutrient broth and incubated for 12, 24, and 48 h at 37°C. Samples of the cultures were collected and divided into two, with one portion heated at 65°C for 30 min to kill residual vegetative cells and the other not heat treated (containing spores and viable vegetative cells). The heat-treated and non-heat-treated samples were serially diluted and plated on BHI agar plates and incubated overnight at 37°C. To determine sporulation efficiency, the number of spores (colonies arising from the heated samples) was divided by the total number of CFU.

Germination assay.

Spore germination assays were performed by the method of Giorno et al. (56). Briefly, purified spores were adjusted to 100 mg/ml in PBS and then diluted in sterile water to an OD₆₀₀ of 0.6. The spores were heat shocked at 65°C for 30 min, and then 100 μl was deposited into wells of 96-well plates (Costar 3370). One hundred microliters of 0.25 mM l-alanine–1 mM inosine was then added as a germinant. Absorbance at 595 nm was then monitored for 30 min using a Biotek Synergy HT plate reader at 37°C with shaking.

Biofilm assay.

Single colonies were inoculated into BHI broth and incubated overnight at 37°C. The cultures were then diluted 1:1,000 in nutrient broth, and 200 μl was deposited into a well of a polystyrene 96-well plates (Costar 3370). The plates were wrapped in Parafilm and incubated at 37°C for 24 h. The wells were washed in sterile water three times and stained with 0.1% (wt/vol) crystal violet. The plates were washed 3 to 5 times in water and allowed to dry. The crystal violet was extracted with 50 μl 40% methanol–10% acetic acid and transferred to a new plate, and the absorbance at 595 nm was measured in a Biotek Synergy HT plate reader.

Protective antigen assay.

Overnight 37°C BHI cultures of each bacterial strain were diluted 1:1,000 into 50 ml nutrient broth supplemented with 0.8% sodium bicarbonate and incubated for 24 h at 37°C under static conditions with an atmosphere of 5% CO₂. Bacterial cells and debris were pelleted by centrifugation at 15,000 rpm for 10 min, and the supernatant was collected and saved. Supernatant proteins were collected by precipitation with 10% trichloroacetic acid (Thermo Fisher). The samples were centrifuged at 15,000 rpm for 10 min at room temperature in a Kompspin KA-18.50 rotor, and the supernatant was discarded. The pellets were resuspended in 20 ml ice-cold acetone and then centrifuged three times at 15,000 rpm for 10 min at room temperature. The resulting pellet was air dried at room temperature. The dried pellets were resuspended in 100 μl of 6 M urea–SDS-PAGE buffer and electrophoresed on 15-well 4-to-20% gradient Mini-protean gels (Bio-Rad) in SDS buffer. For PA, gels were electrophoresed at 190 V until the bromophenol blue dye front ran off the bottom of the gel. The gels were then stained with 0.25% Coomassie R-250 in 50% methanol and 20% acetic acid. For Western blot analysis, membranes were reacted with rabbit anti-protective antigen rabbit polyclonal antiserum (Invitrogen) and 1:20,000 anti-rabbit Ig–HRP conjugate (Invitrogen).

ACKNOWLEDGMENTS

This work was supported by NIAID grant R21AI112725 and the University of Missouri McKee Microbial Pathogenesis endowment fund to GCS. T.M.H. was supported in part by grant DHS-2010-ST-061-AG0001 from the Department of Homeland Security.

Footnotes

Supplemental material is available online only.

Supplemental file 1

Fig. S1 to S4 and Tables S1 to S3. Download JB.00135-21-s0001.pdf, PDF file, 0.7 MB^{(679.8KB, pdf)}

Contributor Information

Charles E. Dann, III, Email: cedann@indiana.edu.

George C. Stewart, Email: stewartgc@missouri.edu.

Michael Y. Galperin, NCBI, NLM, National Institutes of Health

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Fig. S1 to S4 and Tables S1 to S3. Download JB.00135-21-s0001.pdf, PDF file, 0.7 MB^{(679.8KB, pdf)}

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[B56] 56.Giorno R, Mallozzi M, Bozue J, Moody KS, Slack A, Qiu D, Wang R, Friedlander A, Welkos S, Driks A. 2009. Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore. Microbiology (Reading) 155:1133–1145. 10.1099/mic.0.023333-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis

Timothy M Hermanas

Sundharraman Subramanian

Charles E Dann III

George C Stewart

Roles

ABSTRACT

INTRODUCTION

RESULTS

In vivo characterization of individual c-di-GMP-metabolizing enzymes.

FIG 1.

FIG 2.

FIG 3.

Expression of the GGDEF/EAL determinants in B. anthracis Sterne.

FIG 4.

Detection of mCherry fusion proteins on or within B. anthracis spores.

FIG 5.

B. anthracis biofilm production.

Anthrax toxin production.

FIG 6.

BclA incorporation into spores.

Sporulation efficiency.

FIG 7.

The effects of the c-di-GMP system determinants on spore germination efficiencies.

FIG 8.

DISCUSSION

FIG 9.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Identification of the c-di-GMP determinants of B. anthracis.

DNA purification.

Construction of B. subtilis strains expressing the B. anthracis putative c-di-GMP determinants.

B. subtilis swarm expansion and c-di-GMP-responsive riboswitch-GFP reporter assays.

Electroporation of B. anthracis.

Construction of the gene fusions to mCherry.

Generation of B. anthracis deletion mutants.

Construction of complementation plasmids.

Western blot analysis.

Production of spores.

Anti-BclA immunofluorescence and flow cytometry.

Sporulation assay.

Germination assay.

Biofilm assay.

Protective antigen assay.

ACKNOWLEDGMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

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