革兰氏阳性和孢子形成细菌侧孢短芽孢杆菌(Bl)属于短芽孢杆菌系统发育簇。该物种的分离株已证明了对多种无脊椎害虫和植物病害的杀虫效力。两个新西兰分离株,Bl1821L和Bl1951正在开发作为生物农药,用于控制小菜蛾和其他害虫。然而,由于这些地方性分离株的生长经常受到限制,生产可能是一个问题。在之前的工作基础上,据推测,可能涉及推定的噬菌体。在调查生长中断的原因期间,Bl1821L的粗裂解物的电子显微照片显示存在噬菌体\'尾巴样结构。使用具有PEG8000沉淀的软琼脂覆盖方法来区分推定的噬菌体和噬菌体尾巴样结构(细菌素)的拮抗活性。分析测试验证了推定的噬菌体活性的缺乏。使用相同的方法,发现了Bl1821L裂解物对几种革兰氏阳性菌的广谱抗菌活性。蔗糖密度梯度纯化和10kDMWCO浓缩裂解物的SDS-PAGE显示出〜48kD的突出蛋白带,透射电子显微镜显示存在多鞘状结构。〜48kD蛋白的N端测序定位到与芽孢杆菌PBSX噬菌体样元件xkdK具有弱预测氨基酸同源性的基因,其翻译产物与另一个Bl发表的基因组的噬菌体尾鞘蛋白具有90%的氨基酸相似性,LMG15441。生物信息学分析还鉴定了Bl1951基因组中的xkdK同源物。然而,Bl1821L和Bl1951之间的xkdK基因周围区域的基因组比较发现差异,包括两个富含甘氨酸的蛋白质编码基因,它们在Bl1951中含有不完美的重复序列(1700bp),而推定的噬菌体区域位于类似的Bl1821L区域。尽管对具有缺陷噬菌体PBSX的Bl1821L和Bl1951PBSX样区域的基因组组织进行了比较分析,PBSZ,和枯草芽孢杆菌168和W23的PBP180,和芽孢杆菌噬菌体PBP180显示低氨基酸相似性,这些基因以相似的排列编码相似的功能蛋白质,包括噬菌体尾鞘(XkdK),tail(XkdO),霍林(XhlB),和N-乙酰胞壁酰-1-丙氨酸(XlyA)。AMPA分析在Bl1821L的〜48kD测序的蛋白质中鉴定出13个氨基酸的杀菌区段。在测定中纯化的〜48kD噬菌体尾巴样蛋白的拮抗活性与粗裂解物显著不同,在处理后18h,与对照相比,导致Bl1951的活细胞数量减少34.2%。总的来说,鉴定的诱导型噬菌体尾巴样颗粒可能对昆虫病原分离株Bl1821L的体外生长具有影响。
The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.