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United States Department of Agriculture

Agricultural Research Service

Title: ISOLATION AND ANALYSIS OF PEPTIDOGLYCAN FROM BRACHYSPIRA (SERPULINA) PILOSICOLI

Authors
item Trott, Darren
item Stanton, Thaddeus
item Wannemuehler, M - IOWA STATE UNIV.,AMES,IA.

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: October 16, 1999
Publication Date: N/A

Technical Abstract: Bacterial peptidoglycan consists of a glycan backbone with cross-linked peptide chains. It confers shape to a bacterial cell and provides mechanical support. Spirochete peptidoglycan is intimately associated with the outer layer of the cell wall-cytoplasmic membrane complex. It has been isolated from each of the major spirochete genera except for Brachyspira. To assess its suitability as an inner membrane marker, we isolated peptidoglycan from B. pilosicoli, the agent of intestinal spirochetosis. B. pilosicoli strain 95-1000 was treated with Triton X-114, DNAase, RNAase, sodium dodecyl sulfate and proteinase K. Centrifugation and extensive washing resulted in the recovery of a clear gelatinous pellet. Transmission electron micrographs of negatively stained sections of the material showed ghost-like cells that retained their original spirochete helical shape. Amino acid analysis by molar percentage (MP) showed that the purified peptidoglycan consisted of glutamic acid (17.3%), alanine (41.1%), diaminopimelic acid (DAPA) (38%) and trace amounts of methionine (2.7%) and lysine (0.87%). The finding that B. pilosicoli peptidoglycan contains DAPA is surprising as in most genera the major branching diamino amino acid is ornithine. DAPA could be detected at low levels (1.1% MP) in B. pilosicoli total membranes (inner and outer membrane plus flagella). Only trace amounts of DAPA (0-0.3% MP) were detected in purified membrane fractions obtained by osmotic shock and separated by density.

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