Christian-Doppler-Laboratory for Genetically Engineered Lactic Acid Bacteria, Department of Biotechnology, University of Natural Resources and Life Sciences, VIBT, Muthgasse 11, 1190 Vienna, Austria.
Plasmid. 2011 Sep;66(3):159-68. doi: 10.1016/j.plasmid.2011.08.002. Epub 2011 Aug 30.
Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G+C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G+C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.
布赫纳氏乳杆菌可能是最有益的微生物，可有效防止草、玉米和其他植物材料制成的动物饲料青贮饲料有氧变质。其强制性异型发酵特性、耐酸性和健壮性引起了人们对该物种的关注，将其作为青贮饲料起始培养物以及遗传工程应用。首次从同一布赫纳氏乳杆菌（L. buchneri）菌株 L. buchneri CD034 中分离、测序并表征了两个隐性质粒。较大的质粒命名为 pCD034-1，长度为 3424bp，G+C 含量为 38.36%。较小的质粒命名为 pCD034-2，长度为 2707bp，G+C 含量为 38.60%。在两个质粒上我们预测了三个开放阅读框。在 pCD034-1 上，ORF1 编码一个假定的复制蛋白，与来源于植物乳杆菌的 pC194/pUB110 家族质粒的 RepA 蛋白具有 99%的同一性。ORF2 编码一个假定的未知功能蛋白。pCD034-2 的 ORF1 和 ORF2 对应于与来自植物乳杆菌的质粒 pLB4 相似的 RepA 和 RepB 蛋白。两个质粒的 ORF3 编码一个假定的移动蛋白，类似于肠球菌质粒 pF8801。鉴定了双链原点、假定的单链原点和典型的移动起始信号。两个质粒均以相对较高的质粒拷贝数维持。构建了两个携带 pCD034-1 和 pCD034-2 复制起点的穿梭载体，并成功转化了来自同一环境的另外两个物种。因此，我们认为这两个新的布赫纳氏乳杆菌质粒是用于生成 LAB 穿梭和表达载体的有价值资源。
