Molecular Characterization and Proposed Taxonomic Placement of the Biosimulant 'BG' S.A. Burke 1, J.W. Wright 2, M.K. Robinson, B. Bronk 3, and R.L. Warren 1 1 Battelle Dugway Operations 2 U.S. Army Soldier and Biological Chemical Command 3 U.S. Army Dugway Proving Ground burkes@battelle.org
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INTRODUCTION A brief history of the biosimulant BG - Migula describes Bacillus globigii, 1900 - Smith reclassifies, B. subtilis var. niger, 1952 - Gordon eliminates variation designation, 1973 - Nakamura proposes B. atrophaeus sp. nov., 1989 - Fritze and Pukall reclassify ATCC 9372, 2001
INTRODUCTION ATCC 9372 used as a surrogate of B. anthracis by the Dugway Proving Ground Ambiguous status of other pigmented Bacillus strains
INTRODUCTION Amplified Fragment Length Polymorphism (AFLP) - high resolution genotyping technique - use in bacterial taxonomic studies
INTRODUCTION Restriction digest, EcoRI, MseI Adaptor ligation EcoRI fragments EcoRI/MseI fragments MseI fragments Selective amplification, EcoRI-primer*, MseI-primer * * Capillary Electrophoresis
METHODS 16S rrna gene sequencing - MicroSeq 500 (Applied Biosystems) AFLP - 16 selective primer sets - Internal size standard - Data analysis w/ GelCompar II (Applied Maths)
METHODS AFLP - EcoRI +C/MseI +CC, EcoRI +C/MseI +CA - Reproducibility and baseline variability - Removal of ambiguous band classes - Creation of a composite data set of Dice coefficients - UPGMA dendrogram constructed - Bootstrap analysis
A F L P - C C A METHODS 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 A F L P - C C A 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 AFLP-CCA 50.00 80.0 0 100.00 12 0.00 1 40.00 16 0.00 180.00 200.00 220.00 24 0.00 260.00 300.00 32 0.00 35 0.00 3 80.00 450.00 480.00
RESULTS 16S rrna gene sequencing - ATCC 49337 vs 9372 Bacillus 49337 Bacillus 9372 Bacillus 49337 Bacillus 9372 Bacillus 49337 Bacillus 9372 Bacillus 49337 Bacillus 9372
RESULTS 16S rrna gene sequencing - B. atrophaeus vs B. subtilis - 99.3% identity (10 base substitutions in ~1500 bases)
AFLP-CC A+AFLP-CC C AFLP-CCA RESULTS 50.00 60. 0 0 70.00 80.00 90.00 100.00 110.00 120.00 13 0. 0 0 16 0. 0 0 170.00 180.00 190.00 20 0. 0 0 210.00 220.00 230.00 240.00 250.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 520.00 4516 27142 6537 7972 49337 51189 31028 49760 9372 49822 PY79 6633 EcoRI +C/ MseI + CA
RESULTS 4516 27142 6537 7972 49337 51189 31028 49760 9372 49822 PY 79 6633 AFLP-CC A+AFLP-CC C AFLP-CCC 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 160.00 170.00 180.00 190.00 200.00 210.00 220.00 230.00 240.00 250.00 260.00 280.00 300.00 320.00 340.00 350.00 360.00 380.00 400.00 420.00 440.00 460.00 480.00 500.00 EcoRI +C/ MseI +CC
RESULTS AFLP - total number of fragments = 164 - total among pigmented Bacilli = 72-41 common fragments - 31 polymorphisms
All AFLP RESULTS 20 30 40 50 60 70 80 90 100 55 80 100 100 72 100 10 0 10 0 10 0 100 B. circulans 4516 B. pumilis 27142 B. atrophaeus 6537 B. atrophaeus 7972 B. atrophaeus 49337 T B. atrophaeus 51189 B. subtilis 31028 B. subtilis 49760 B. atrophaeus 9372 B. subtilis 49822 B. subtilis PY79 B. subtilis 6633
SUMMARY AFLP data were useful in discriminating between closely related taxa of Bacillus Species level variation was easily detected with AFLP A distinct cluster of B. atrophaeus isolates was revealed AFLP data indicate a clear difference between B. atrophaeus type strain and Dugway BG A subspecies of B. atrophaeus is proposed; B. atrophaeus subsp. globigii Dugway BG is B. atrophaeus subsp. globigii