Miljkovic Marija, Strahinic Ivana, Tolinacki Maja, Zivkovic Milica, Kojic Snezana, Golic Natasa, Kojic Milan
Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444/a, P.O. Box 23, 11010, Belgrade, Serbia.
Laboratory for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444/a, P.O. Box 23, 11010, Belgrade, Serbia.
PLoS One. 2015 May 8;10(5):e0126387. doi: 10.1371/journal.pone.0126387. eCollection 2015.
Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.
从实验室保藏的菌株中挑选出11株具有强聚集能力的乳酸杆菌菌株。其中两株菌株中，与聚集能力相关的基因位于质粒上，且发现与胶原结合密切相关。使用新型广宿主穿梭克隆载体pAZILSJ克隆了副干酪乳杆菌副干酪亚种BGNJ1-64的自聚集促进蛋白（AggLb）编码基因。从SacI质粒文库中筛选出含有11377 bp DNA片段的克隆pALb35，因其能赋予载体聚集表型。对完整片段进行测序，检测到4个潜在的开放阅读框，包括aggLb基因和3个周围的转座酶基因。AggLb是已知乳酸杆菌中最大的细胞表面蛋白，由2998个氨基酸（318,611 Da）组成。AggLb属于胶原结合超家族，其C端区域包含20个连续重复序列，甚至在核苷酸水平上也是相同的。缺失aggLb会导致细胞聚集能力丧失，而过表达则会增加细胞聚集、疏水性和胶原结合潜力。基于BGNJ1-64的aggLb基因用三组引物进行PCR筛选，能够在11株选定的聚集阳性乳酸杆菌菌株中的5株中检测到同一类型的aggLb基因。aggLb的异源表达证实了AggLb蛋白在细胞聚集和特异性胶原结合中的关键作用，表明AggLb在宿主组织的有效定殖和预防病原体定殖方面具有有益的益生菌功能。
