Molecular Characterization and Proposed Taxonomic Placement of the Biosimulant 'BG'
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1 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
2 Report Documentation Page Form Approved OMB No Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE 01 OCT REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Molecular Characterization and Proposed Taxonomic Placement of the Biosimulant BG 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Battelle Dugway Operations 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 11. SPONSOR/MONITOR S REPORT NUMBER(S) 13. SUPPLEMENTARY NOTES See also ADM001851, Proceedings of the 2003 Joint Service Scientific Conference on Chemical & Biological Defense Research, November 2003., The original document contains color images. 14. ABSTRACT 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT UU a. REPORT unclassified b. ABSTRACT unclassified c. THIS PAGE unclassified 18. NUMBER OF PAGES 15 19a. NAME OF RESPONSIBLE PERSON Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18
3 INTRODUCTION A brief history of the biosimulant BG - Migula describes Bacillus globigii, Smith reclassifies, B. subtilis var. niger, Gordon eliminates variation designation, Nakamura proposes B. atrophaeus sp. nov., Fritze and Pukall reclassify ATCC 9372, 2001
4 INTRODUCTION ATCC 9372 used as a surrogate of B. anthracis by the Dugway Proving Ground Ambiguous status of other pigmented Bacillus strains
5 INTRODUCTION Amplified Fragment Length Polymorphism (AFLP) - high resolution genotyping technique - use in bacterial taxonomic studies
6 INTRODUCTION Restriction digest, EcoRI, MseI Adaptor ligation EcoRI fragments EcoRI/MseI fragments MseI fragments Selective amplification, EcoRI-primer*, MseI-primer * * Capillary Electrophoresis
7 METHODS 16S rrna gene sequencing - MicroSeq 500 (Applied Biosystems) AFLP - 16 selective primer sets - Internal size standard - Data analysis w/ GelCompar II (Applied Maths)
8 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
9 A F L P - C C A METHODS A F L P - C C A AFLP-CCA
10 RESULTS 16S rrna gene sequencing - ATCC vs 9372 Bacillus Bacillus 9372 Bacillus Bacillus 9372 Bacillus Bacillus 9372 Bacillus Bacillus 9372
11 RESULTS 16S rrna gene sequencing - B. atrophaeus vs B. subtilis % identity (10 base substitutions in ~1500 bases)
12 AFLP-CC A+AFLP-CC C AFLP-CCA RESULTS PY EcoRI +C/ MseI + CA
13 RESULTS PY AFLP-CC A+AFLP-CC C AFLP-CCC EcoRI +C/ MseI +CC
14 RESULTS AFLP - total number of fragments = total among pigmented Bacilli = common fragments - 31 polymorphisms
15 All AFLP RESULTS B. circulans 4516 B. pumilis B. atrophaeus 6537 B. atrophaeus 7972 B. atrophaeus T B. atrophaeus B. subtilis B. subtilis B. atrophaeus 9372 B. subtilis B. subtilis PY79 B. subtilis 6633
16 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
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