Guzman Juan, Raval Dipansi, Hauck Dirk, Titz Alexander, Poehlein Anja, Degenkolb Thomas, Daniel Rolf, Vilcinskas Andreas
Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology, Giessen, Germany.
Institute for Insect Biotechnology, Justus-Liebig-University of Giessen, Giessen, Germany.
Access Microbiol. 2023 Sep 22;5(9). doi: 10.1099/acmi.0.000647.v4. eCollection 2023.
Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro--acetylmuramic acid (1,6-anhydro-MurNAc). The gene from was amplified and cloned into a pET21b expression vector and the protein was expressed in BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla and . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.
休眠细菌细胞不分裂且不能立即培养，但它们以低代谢活性状态持续存在，这种生理状态具有临床相关性，例如在潜伏性结核病中。复苏促进因子（Rpfs）是作为介导生长和复制的信号分子的蛋白质。在本研究中，我们旨在使用昆虫和土壤样本测试来自[具体来源未提及]的Rpfs对培养细菌数量和多样性的影响，并检查是否可以用Rpf的推定反应产物1,6-脱水-N-乙酰胞壁酸（1,6-anhydro-MurNAc）重现可培养性的增加。从[具体来源未提及]扩增[具体基因未提及]基因并克隆到pET21b表达载体中，该蛋白质在大肠杆菌BL21(DE3)细胞中表达，并使用六组氨酸标签通过亲和色谱法纯化。1,6-脱水-N-乙酰胞壁酸使用报道的化学合成方法制备。将重组Rpf蛋白或1,6-脱水-N-乙酰胞壁酸添加到R2A培养基中，并检查它们对来自八个环境样本（包括四个蟑螂肠道和四个土壤）中细菌可培养性的影响。对所有八个样本进行三种不同处理：Rpf、1,6-脱水-N-乙酰胞壁酸或空白对照，测量菌落形成单位、16S rRNA基因拷贝数以及16S rRNA基因的Illumina扩增子测序。尽管蛋白质更有效，但Rpf和1,6-脱水-N-乙酰胞壁酸均增加了样本中的菌落形成单位数量和16S rRNA基因拷贝数。Rpf和1,6-脱水-N-乙酰胞壁酸促进了多种细菌的培养，特别是厚壁菌门和变形菌门的某些进化枝。本研究为改进培养策略开辟了道路，旨在分离和研究尚未描述的活细菌生物。
