Supporting Information
“The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-mediated Ecological Adaptations”
Raymond Schuch and Vincent A. Fischetti
Supplemental Tables S1-S7
Supplemental Figure Legend S1-S8
Supplemental Figures S1-S8
Table S1. Phenotypic and genotypic analysis of B. anthracis and B. cereus strains.
Strain / Source / Hem / FosR / BaCHR / BaVIR / PlyGBD / Biofilm / / GroupB.anthracis
Sterne / Lab strain / - / + / + / - / + / - / no
Sterne / Lab strain / - / + / + / + / + / / 20*
B. cereus
T / Lab strain / + / - / - / - / - / - / no
ATCC 4342 / Lab strain / + / - / - / - / + / - / no
ATCC 25621 / Cow dung / - / - / - / - / + / / 415
Environ. strains
RS1615 / Soil / - / + / + / + / + / / 1615 / anthracis
RS1046 / Worm gut / - / + / + / - / + / + / Wip5 / anthracis
RS1255 / Human tonsil / + / - / - / - / - / + / Htp1 / cereus
RS1045 / Worm gut / + / + / - / - / + / + / Wip4 / cereus
RS1557 / Rhizosphere / + / + / - / - / - / + / Frp2 / cereus
RS1047 / Worm gut / + / + / - / - / - / / 1047 / cereus
RS421 / Worm gut / + / n.d. / - / - / - / / 421 / cereus
RS423 / Worm gut / - / n.d. / - / - / - / / 423 / cereus
Environmental strains identified in this study were compared to known B. anthracis and B. cereus isolates in the following phenotypic and genotypic analyses: Hem, -hemolysis on blood agar plates; FosR, growth in culture supplemented with 150 mg fosfomycin ml-1; BaCHR, PCR-positive for B. anthracis-specific chromosomal loci at the four native,non-inducible prophage sequences and the diagnostic marker Ceb-Bams 30; BaVIR, PCR-positive for the B. anthracis virulence plasmid loci; PlyGBD, binding to GFP-PlyGBD; Biofilm, formation of multicellular structures at the liquid-air interface of 10 ml LD cultures incubated for 2 months at room temperature; , the name given to an inducible B. anthracis-infective phage identified in that strain (“no” indicates that no phage was observed); Group, indicates a preliminary taxonomic assignment based on the findings in this work. *, denotes the fact that 20 has been described in previous work. “n.d.” indicates that the experiment was not done.
Table S2. The loss of phage during growth in BHI medium.
Strain / Frequency of phage-cured derivativesSterne+Wip1 / <1x10-4
Sterne+Wip2 / <1x10-4
Sterne+Wip4 / 8x10-4
Sterne+Wip5 / <1x10-4
Sterne+Frp2 / <1x10-4
Sterne+Bcp1 / 9x10-4
Overnight BHI cultures were plated on BHI agar and resulting phage-cured derivatives were identified based on colony morphology and confirmed by PCR analysis using phage-specific primers. “<” indicates that curing was below the threshold of detection. These experiments were performed in duplicate and all values represent averages.
Table S3. Bcp1 adsorption characteristics.
Strain / Bound phage at 5 minutes (%) / Bound phage at 60 minutes (%)B. cereus T / 95 / 96
B. anthracis Sterne / 28 / 45
B. anthracis Sterne / 17 / 52
B. cereus ATCC 10987 / 0 / 0
Assays were performed by mixing 1-5 x 103 phage particles with ~1x106 mid-log phase bacteria for 5 or 60 minutes at 37°C with aeration. After centrifugation, supernatants were removed, sterilized, and titered using B. cereus T as the host. The percentage of adsorption was calculated as follows: [1-(initial phage titer/post-adsorption titer)]x100. The experiments were all performed in triplicate.
Table S4. Effect of lysogeny on the sporulation (Spo) phenotype of B. anthracis Sterne.
Infecting phage / Bacteria ml-1 / Spo- bacteria ml-1 / Frequency of Spo+ lysogens (%) / Frequency of Spo- lysogens (%)W / 1.0 ± 0.1 x 109 / <4.0 x 105 / 18.4 / <0.04
Wip2 / 7.7 ± 0.9 x 108 / <4.0 x 105 / 15.3 / <0.05
Wip4 / 8.7 ± 1.4 x 108 / 3.2 ± 1.2 x108 / <0.4 / 36.7
Wip5 / 5.7 ± 0.9 x 108 / 2.5 ± 0.4 x108 / <0.7 / 43.8
Frp1 / 6.7 ± 0.5 x 108 / 1.6 ± 0.4 x108 / <0.5 / 23.8
Frp1 (heat-killed) / 6.2 ± 0.4 x 108 / <4.0 x 105 / <0.6 / <0.06
No phage / 7.1 ± 0.9 x 108 / <4.0 x 105 / n.a. / n.a.
Cultures were infected (MOI=0.5) for 30 minutes, washed, and plated at various dilutions on BHI agar. Resulting bacterial colonies were examined for colony morphology and enumerated. Large, opaque colonies are Spo-, while small, matte colonies are Spo+. Spo phenotypes were confirmed by microscopic analysis of colonies subcultured on LD sporulation agar. Stable lysogens were confirmed by PCR with phage-specific primers. As a control, heat-killed Frp1 samples were incubated at 65°C for 30 min prior to infection to destroy its infective capacity. The frequency of lysogenization among infected cells [(stable lysogens/total number of cells) X 100] was determined and is shown. Threshold levels for detecting Spo+ and Spo- lysogens were 4 x105 and 4 x106 bacteria ml-1, respectively. Experiments were performed in triplicate. “n.a.” indicates that the experiment was not applicable here.
Table S5. Frequency of B. anthracis derivatives infected with phages that are shed by lysogens during growth in different media types.
Phage donor (lysogen) / Sheep Blood / BHI / LDSterne/W / 0.027 / <1.5x10-4 / <7.6x10-5
Sterne/Wip1 / 0.016 / <1.6x10-4 / <5.0x10-4
Sterne/Bcp1 / <1.6x10-4 / 0.02 / 0.001
Sterne/Wip4 / <1.5x10-4 / 0.3 / 0.02
Overnight cultures of B. anthracis Sterne/pASD2 (kanamycin- and spectinomycin-resistant) were co-inoculated with indicated lysogens into one of three types of growth media and incubated for 2 days at 30°C with aeration. Culture aliquots and dilutions thereof were plated on BHI agar supplemented with antibiotics to select for Sterne/pASD2 survivors and against the phage donor strains. Infection of Sterne/pASD2 by shed phages was determined by PCR analysis with phage-specific primers of >200 Sterne/pASD2 colonies resulting from each condition. The reported frequency was determined based on the number of PCR-positive derivatives per ml of culture/the total number of Sterne/pASD2 bacteria per ml of culture. “<” indicates that stable infection, if any, was below the threshold of detection. These experiments were performed in duplicate and all values represent averages.
Table S6. Bacterial strains and plasmids used in this study.
Strain or plasmid / Genotype and description1 / Reference or sourceB. anthracis
Sterne / pX01+ strain 34F2 / J. Lederberg
Sterne / Plasmid-cured 34F2 / J. Lederberg
Sterne::Wip4 BA3443 / BA3443 insertional mutant / This study
Sterne::Wip4 BA4109 / BA4109 insertional mutant / This study
Sterne::Wip4BA0672 / BA0672 insertional mutant / This study
Sterne::Wip4BA1295 / BA1295 insertional mutant / This study
B. cereus
ATCC 14579 / Reference strain / ATCC
ATCC 25621 / Cow dung isolate / ATCC
T / Laboratory strain / A.Aronson
ATCC 11950 / Source of W phage / [46]
Plasmids
pBAD24 / Arabinose-inducible E. coli expression vector / [98]
pBAD24::gfp-plyGBD / Production of GFP-PlyGBD / [46]
pASD2 / KanR SpcR AmpR E. coli-B. anthracis shuttle vector / [82]
pASD2::gfp / gfpmut2 vector / This study
pASD2::PBA3443-gfp / BA3443 promoter-gfpmut2 fusion vector / This study
pASD2::PBA0672-gfp / BA0672 promoter-gfpmut2 fusion vector / This study
pASD2::PBA1295-gfp / BA1295 promoter-gfpmut2 fusion vector / This study
pASD2::PBA3436-gfp / BA3436 promoter-gfpmut2 fusion vector / This study
pASD2::BA3443 / BA3443 mutagenesis vector / This study
pASD2::BA4109 / BA4109 mutagenesis vector / This study
pASD2::BA0672 / BA0672 mutagenesis vector / This study
pASD2::BA1295 / BA1295 mutagenesis vector / This study
pASD2::bcp25 / bcp25 mutagenesis vector / This study
pASD2::wip39 / wip39 mutagenesis vector / This study
pASD2::P-bcp25,26 / Fragment bearing bcp25,26 locus with 353-bp promoter / This study
pASD2::P-wip38,39 / Fragment bearing wip38,39 locus with 258-bp promoter / This study
pASD2::bcp25,26 / Fragment bearing bcp25,26 locus with no promoter / This study
pASD2::wip38,39 / Fragment bearing wip38,39 locus with no promoter / This study
pWH1520 / TcR AmpR E. coli-Bacillus shuttle vector / MoBiTec, Inc.
pWH1520::bcp25,26 / Fragment bearing bcp25,26 locus with no promoter / This study
pWH1520::wip38,39 / Fragment bearing wip38,39 locus with no promoter / This study
1Kan, kanamycin; Spc,spectinomycin; Tc, tetracycline; Amp, ampicillin.
2American Type Culture Collection (Manassas, VA).
Table S7. Select primers used in this study.
Name / Sequence / FunctionWip1 up / 5’-actggaaaacaccctgac-3’ / Confirm lysogeny
Wip1 down / 5’-gtggttcaaatgcggttct-3’ / Confirm lysogeny
Wip2 up / 5’-gcttctgcgttctgatttaatctg-3’ / Confirm lysogeny
Wip2 down / 5’-ggtcaaaagtatacattcgttgttaaagg-3’ / Confirm lysogeny
Wip4 up / 5’-cgaattacttttccgagtggcaatg-3’ / Confirm lysogeny
Wip4 down / 5’-ccccatcacttggcgctttc-3’ / Confirm lysogeny
Wip5 up / 5’-caaattctgttttatcagaccgcttctg-3’ / Confirm lysogeny
Wip5 down / 5’-ctccactcaatttcactatgaaaatcg-3’ / Confirm lysogeny
Slp1 up / 5’-gtagaaggtgtagcttatattgaagg-3’ / Confirm lysogeny
Slp1 down / 5’-ctattgtcaatggtggatcattgg-3’ / Confirm lysogeny
Bcp1 up / 5’-atgggttacatcgttgatatg-3’ / Confirm lysogeny
Bcp1 down / 5’-ttacttgaatgtgccccaag-3’ / Confirm lysogeny
W up / 5’-atgagaattgcgctttacag-3’ / Confirm lysogeny
W down / 5’-ttatacgacaatcccactgtt-3’ / Confirm lysogeny
bcp25,26 pro up / 5’-ctcccgggaattcgtagctaaatgcggttttagatta-3’ / Expression clone
bcp25,26 pro down / 5’-ctcccgggaatttaatagctgcttgctttttgctttcaaatg-3’ / Expression clone
bcp25,26 up / 5’-ctcccgggatgaataagaaaagggcaggtagcac-3’ / Expression clone
bcp25,26 down / 5’-ctcccgggttaaactcctcctagttctaatttaatt-3’ / Expression clone
wip38,39 pro up / 5’-ctgatatcaattatggcattactcatgaaatgtgatg-3’ / Expression clone
wip38,39 pro down / 5’-ctgatatcaattctactttctcctttagcaaatct-3’ / Expression clone
wip38,39 up / 5’-ctgatatcatggctaataataaaaacggggaac-3’ / Expression clone
wip38,39 down / 5’-ctgatatctcaatcttgaagacctccttctttacc-3’ / Expression clone
BA3443 pro up / 5’-ctgaattcaattggaaacggattgcgcaaatgcc-3’ / Promoter-gfp fusion
BA3443 pro down / 5’-ctgaattcaatttcttttacatttgttcgttcgtc-3’ / Promoter-gfp fusion
BA0672 pro up / 5’-ctgaattcaattgctccaaacgttgttgtaaaggttc-3’ / Promoter-gfp fusion
BA0672 pro down / 5’-ctgaattcaattggtgttgtagatacattttctttc-3’ / Promoter-gfp fusion
BA1295 pro up / 5’-ctgaattcaattgtgatatactcgtatgctaactatg-3’ / Promoter-gfp fusion
BA1295 pro down / 5’-ctgaattcaattctgataaatcaagaccgctatctacag-3’ / Promoter-gfp fusion
BA3436 pro up / 5’-ctgaattcaatttggatattgagcattttatataaag-3’ / Promoter-gfp fusion
BA3436 pro down / 5’-ctgaattcaattttcaatcggaatgtggactcc-3’ / Promoter-gfp fusion
gfpmut2 up / 5’-gatggtaccgaattctaagaaggagatatacatatgagtaaagg-3’ / Promoter-gfp fusion
gfpmut2 down / 5’-catgatatccttatttgtatagttcatccatgccatg-3’ / Promoter-gfp fusion
BA3443KO1 / 5’-acaggtacccatgctgacgaacgaacaaatgta-3’ / mutagenesis
BA3443KO2 / 5’-acaggtaccaatcggaagaggtttactagcactt-3’ / mutagenesis
BA0672KO1 / 5’-acaggtaccatgagtgctccattagcgtacg-3’ / mutagenesis
BA0672KO2 / 5’-acaggtaccctttagatgttggaacttgtccattca-3’ / mutagenesis
BA1295KO1 / 5’-acaggtaccccagtctgtatatgctgaaacgc-3’ / mutagenesis
BA1295KO2 / 5’-acaggtacctaccatttagaccacctctaactg-3’ / mutagenesis
BA4109KO1 / 5’-acaggtaccttagtaaaaggtagaaaattagcg-3’ / mutagenesis
BA4109KO2 / 5’-acaggtaccctcaaacttatggccatctggatct-3’ / mutagenesis
Bcp25KO1 / 5’-cattagtcgaacacaacgctcgacttg-3’ / mutagenesis
Bcp25KO2 / 5’-gaagttctgttcttcaagtagttgcc-3’ / mutagenesis
Wip39KO1 / 5’-gataaagaacggactgctgtagcttgg-3’ / mutagenesis
Wip39KO2 / 5’-agttgtattccagataggctatgtcc-3’ / mutagenesis
PlyG BD1 / 5’-acagatatcattcagaaggttaagaatgg-3’ / GFP-PlyGBD fusion
PlyG BD2 / 5’-cccaagcttttatttaacttcataccacc-3’ / GFP-PlyGBD fusion
bcp25-1 up / 5’-gatacttcaaaaggactacagttttcc-3’ / RT-PCR
bcp25-2 down / 5’-cgtactttaatgtaagtacggattg-3’ / RT-PCR
bcp25-3 mid / 5’-cggtacatacgcaaataagatatctc-3’ / RT-PCR
bcp25-4 up / 5’-cgatggattgacgcagagattggcaac-3’ / RT-PCR
bcp25-5 down / 5’-cgtaaaatgctaaacctgagaaccagtc-3’ / RT-PCR
bcp26-1 up / 5’-tacgcattcagtacgtacgcttatc-3’ / RT-PCR
bcp26-2 down / 5’-cctttagaatgtgctcacagaac-3’ / RT-PCR
bcp26-3 mid / 5’-ccttcttgaaacatgtcttcgaactcg-3’ / RT-PCR
bcp26-4 up / 5’-gatatcgactcaggttacgcattcagtac-3’ / RT-PCR
bcp26-5 down / 5’-caccacctccttcaatgttgcgg-3’ / RT-PCR
wip38-1 up / 5’-atggctaataataaaaacggg-3’ / RT-PCR
wip38-2 down / 5’-ccaaactgagttaaatcttcaatatc-3’ / RT-PCR
wip38-3 up / 5’-gcactttcctaagtttgataaaggagag-3’ / RT-PCR
wip38-4 down / 5’-ccgataattttaggaacagcatatg-3 / RT-PCR
wip39-1 up / 5’-cctgaattcggatgtgcattttcaac-3’ / RT-PCR
wip39-2 down / 5’-cagctttaaattctcttattac-3’ / RT-PCR
wip39-3 up / 5’-ccctaaagtatcaaccaagtac-3’ / RT-PCR
wip39-4 down / 5’-gagcagctctttgattaatctttgc-3’ / RT-PCR
sap-1 up / 5’-gctggcgttattaaaggtacagg-3’ / RT-PCR
sap-2 down / 5’-atcgctttgttagttactttgat-3’ / RT-PCR
sigF-1 up / 5’-accgcggatacgaaccagacg-3’ / RT-PCR
sigF-2 down / 5’-gatcaaattgcagatcaatctga-3’ / RT-PCR
Figure S1. The appearance of asporogenous (Spo-) B. anthracis lysogens. (A) A Spo- Wip4 lysogen (indicated by arrow) appearing in field of Spo+ non-lysogens. Here, B anthracis strain Sterne was infected with Wip4 (MOI of 0.01) for 16 hours in BHI liquid culture and plated for 24 hours on soil-extract agar. The indicated colony is ~1-2 mm in diameter. (B) A Spo- Frp1 lysogen (indicated by arrow) in field of Spo+ non-lysogens. Indicated colony is 4 mm in diameter. (C) The appearance of Spo- derivatives of Sterne in Wip4-infected cultures. Here, mid-log phase liquid BHI cultures were infected with a range of phages concentrations (MOIs) for 3 hours, washed and plated for 16 hours on BHI. Resulting colonies were screened by PCR with phage-specific primers to identify lysogens. All lysogens corresponded to Spo- colonies. Numbers are mean averages (n=10) of Spo- lysogens appearing in each condition and the error bars are standard deviations.
Figure S2. Bcp1 adsorbs to B. anthracis Sterne. Bacteria were infected with Bcp1 at an MOI of 1 (A) or 50 (B) for 15 minutes at 37°C, washed twice with PBS, fixed, and analyzed by thin-section electron microscopy. Scale bars are 50 nm. Arrows indicate phage heads that are either free (A) or full (B) of the Bcp1 genome. The absence of DNA in the phage head suggests that the genome translocated into Sterne.
Figure S3. Biofilms formed by B. anthracis and environmental B. cereus strains. Either settled material (for Sterne) or biofilms (for Sterne/Wip1, RS423, and RS421) formed at the liquid-air interface of 3 month-old BHI cultures grown without aeration at 24°C were recovered, labeled with GFP-PlyGBD, and examined by phase-constrast and fluorescence microscopy at 200X and 2000X magnifications. Exposure times are indicated for fluorescence images. (A) The biofilms of Sterne/Wip1 consist of a matrix enriched with the B. anthracis exopolysaccharide (the binding target of GFP-PlyGBD). Three distinct regions are observed in 2000X images, including spore/vegetative mixtures, vegetative-enriched, and spore-enriched zones from left to right. (B) Settled material in 3 month Sterne cultures consists predominantly of cellular debris that does not bind well to GFP-PlyGBD. Sterne alone does not form biolfims, thus only the settled material was analyzed. (C) Biofilms formed by RS423, a B. cereus s.l. strain from the worm gut, are in a GFP-PlyGBD-labeled matrix. (D) Biofilms formed by RS421, a B. cereus s.l. strain from the worm gut, are in a GFP-PlyGBD-labeled matrix.
Figure S4. Amino acid sequence alignment of known and putative bacterial sigma factors. Identical residues are highlighted by black backgrounds. Conserved amino acid changes are highlighted by gray backgrounds. SigF is the B. anthracis sigma factor, F, encoded by BA4294. The alignment was generated by ClustalW and displayed using the BOXSHADE program.
Figure S5. RT-PCR analysis of bcp25,26 and wip38,39 mRNA. We extracted mRNA from Sterne derivatives grown for 3 hours at 37°C in LD (A-D) or BHI (E) medium. The mRNA samples incubated either with (+) or without (-) reverse transcriptase (RT). Resulting cDNA was analyzed with primers listed to the left of each panel (sequences are in Table S6). Lane M is the 1 Kb Plus DNA ladder (New England Biolabs). Sizes in base pairs are at the right of each gel. (A) The Sterne/Bcp1 lyosgen. Amplifications were performed with primers bcp25-1,2 (lanes 1, 3 and 5) or bcp26-1,2 (lanes 2, 4, and 6) using either genomic DNA (lanes 3 and 4) or RT-treated (lanes 1 and 2) and untreated (lanes 5 and 6) mRNA. As controls, primers for sap (BA0885, a locus expressed during vegetative growth) and sigF (BA4294, a locus expressed only during sporulation) were used. (B) The Sterne/Wip4 lyosgen. Amplifications were performed with primers wip38-1,2 (lanes 1 and 3) or wip39-1,2 (lanes 2 and 4) using RT-treated (lanes 1 and 2) and untreated (lanes 5 and 6) mRNA. The wip38-1,2 primers span the intergenic region of wip38 and wip39. (C) Analysis of Sterne/pASD2::P-wip38,39 (Lanes 1 and 3) or Sterne/pASD2::wip38,39PROMOTERLESS (lanes 2 and 4). The RT-treated samples were amplified with primers wip38-3,4 (lanes 1 and 2) or wip39-3,4 (lanes 3 and 4). The wip38-3,4 primers span the intergenic region of wip38 and wip39. (D) Analysis of Sterne/pASD2::P-bcp25,26 (Lanes 1, 3, and 5) or Sterne/pASD2::bcp25,26PROMOTERLESS (lanes 2, 4, 6). Amplifications were performed on RT-treated samples using primers bcp25-4,5 (lanes 1 and 2), bcp26-4,5 (lanes 3 and 4), or bcp25-3,bcp26-3 which span the bcp25-bcp26 intergenic region (lanes 5 and 6). E) Analysis Sterne lysogens during vegetative growth in BHI. Amplifications were performed on RT-treated samples from Sterne/Bcp1 (with bcp25-1,2 and bcp26-1,2 primers) and Sterne/Wip4 (with wip38-1,2 and wip39-1,2 primers).
Figure S6. Expanded analysis of survival in the soil and earthworm. Survival at indicated times after inoculation (solid lines) is shown as CFUs per gram of recovered soil or worm guts. Similarly, shedding of free phages (dashed lines) is shown as PFUs extracted per gram of soil or worm guts. Data is shown for B. anthracis viability (vegetative cells and spores; squares), spores alone (diamonds), and free phages (triangles). Values are reported as mean averages (n=5) and error bars are standard deviations. (A) Soil survival for Sterne/pASD2 lysogens. (B) Earthworm gut survival for Sterne/pASD2 lysogens. (C) Survival of environmental B.cereus strain RS1045 and its phage-cured derivative (RS1045CURED) in the soil and earthworm gut. (D) Survival of environmental B. anthracis strain RS1046 and its phage-cured derivative (RS1046CURED) in the soil and earthworm gut. (E) Infection of B. anthracis during co-culture with lysogens in soil microcosms. Strains Sterne/pASD2 and Sterne/pASD2 were either inoculated alone or with B. cereus RS1045 or B. anthracis RS1046. At the indicated time points, Sterne/pASD2 and Sterne/pASD2 were selectively recovered and scored by PCR for infection with either Wip4 (the phage shed by RS1045) or Wip5 (the phage shed by RS1046). Survival of Sterne/pASD2 and Sterne/pASD2 inoculated alone (squares) and their derivatives that have become stably infected with Wip4 (closed circles) or Wip5 (open circles) are shown.
Figure S7. Microscopic analysis of B. anthracis strains recovered from soil microcosms. Culture aliquots of indicated strains were removed at 3 months, labeled with GFP-PlyGBD, and analyzed. Phase-contrast and corresponding fluorescence images are shown at 200X and 2000X magnification. The exposure time for each image was 0.3 seconds.
Figure S8. The shedding of bacteriophage by B. anthracis and its lysogens. B. anthracis strain Sterne, its indicated lysogens, and the environmental B. anthracis strain RS1615 were examined. Numbers are mean averages (n=5) of PFUs shed into the media during the culture of each stain and the error bars are standard deviations.