Kaplan Jeffrey B, Meyenhofer Markus F, Fine Daniel H
Department of Oral Biology, New Jersey Dental School Electron Microscopy Facility, New Jersey Medical School, Newark, New Jersey 07103, USA.
J Bacteriol. 2003 Feb;185(4):1399-404. doi: 10.1128/JB.185.4.1399-1404.2003.
The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.
革兰氏阴性口腔细菌伴放线放线杆菌被认为是人类多种形式牙周病的病原体。在肉汤中培养时，伴放线放线杆菌的新鲜临床分离株会在玻璃、塑料和唾液包被的羟基磷灰石等表面形成坚韧的生物膜，这一特性可能在该细菌定殖于口腔并引发疾病的能力中发挥重要作用。我们通过光学显微镜、扫描电子显微镜和透射电子显微镜检查了在载玻片和聚苯乙烯培养皿上生长的伴放线放线杆菌生物膜菌落的形态。我们发现，伴放线放线杆菌形成了不对称的、有叶状的生物膜菌落，这些菌落呈现出复杂的结构特征，包括菌落在外侧有一层紧密堆积的细胞，而内侧有未聚集的细胞以及大的透明腔隙。成熟的生物膜菌落会将单个细胞或小细胞簇释放到培养基中。这些释放出的细胞附着在培养容器表面并形成新的菌落，从而使生物膜得以扩散。我们分离出三个转座子插入突变体，它们产生的生物膜菌落缺乏内部未聚集的细胞，并且无法将细胞释放到培养基中。所有三个转座子插入都定位到脂多糖O多糖（O-PS）成分合成所需的基因上。携带互补野生型基因的质粒恢复了突变菌株合成O-PS并将细胞释放到培养基中的能力。我们的研究结果表明，伴放线放线杆菌生物膜的生长和脱离是离散的过程，并且生物膜细胞的脱离显然涉及生物膜菌落在内部形成注定要从菌落中释放的未聚集细胞。
