Siderophore production and differentiation of Eubacterium yurii subspecies yurii, margaretiae, and schtitka

Abstract

Eubacterium yurii (Ey) subsp. yurii (Eyy), margaretiae (Eym) and schtitka (Eys), the "test-tube brush" (TTB) bacteria, are gram-positive non-sporeforming anaerobic rods originally isolated from the gingival sulci of patients with periodontitis. The objectives of this study were: (1) to determine whether Ey subspp. could be differentiated based on cellular fatty acid composition (CFA), enzyme profile, the presence of bacteriophage and/or large plasmids, and the Congo red/hemin binding tendencies; (2) to determine whether Ey produced any of the common siderophores: catechols, hydroxymates and citrates, as well as iron regulated proteins. Anaerobes have heretofore been dismissed as siderophore producers because it has been speculated that their environment allows reduction of ferric to ferrous ions. Ey was differentiated from other oral Eubacterium based on the CFA. Using An-Ident, Eyy was separated from the other two subspecies by phosphatase activity. MitomycinC inducible bacteriophages were observed in both Eyy and Eym by electron microscopy. No large plasmids were found in any Ey subspp. as determined by an alkaline heat lysis method. Congo red/hemin binding in Eys and Eym were similar. EDTA flocculation test could be used to distinguish Eys from Eym. Production of siderophores was demonstrated by thin layer chromotography, spectrophotometric assays and by the use of CAS reagent. Eym were shown to produce hydroxymates and increase in catechols and citrate compounds when grown in trypticase soy broth (TSB) supplemented with an iron chelator {dollar}2\sp\prime2\sp\prime{dollar} dipyridyl (DIP). SDS-PAGE protein profiles of whole cell lysates of Eym grown in the presence of DIP demonstrated an increase in protein bands at 97.4, 54 and 42 kDa. In response to iron chelation during growth of Eym, catechol and citrate compounds were reduced initially while hydroxymates were doubled. The Bio-Rad Rotofor fractionator could be used to concentrate the siderophores into fractions. Further studies will be needed to isolate and characterize the Ey siderophores, and to determine whether other bacteria in the periodontal pocket could benefit from these siderophores.