Version 3: The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria

Bacterial dormant 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 (Rpf) are proteins that act as signaling molecules mediating growth and replication. In this study we aimed to test the effect of Rpf  from Micrococcus luteus 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-MurNAc. The rpf gene from M. luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in E. coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-anhydro-N-acetylmuramic acid (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 examined on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils. 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 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 specially certain clades of the phyla Actinomycetota and Bacillota. This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.