Effect Of Resuscitation Promoting Factor On The Culturability Of Freshwater Bacterioplankton

Generally, only a small fraction of the bacteria present in natural samples are culturable in laboratory media. The cultivation efficiency is usually below 1%. Most bacterial cells are in a state called Viable but Nonculturable (VBNC). Possible reason is lack of cell-to-cell communication in laboratory media. Microorganism is a most important resource of bioactive compounds. The low cultivation efficiency, however, become one of the bottlenecks to screen new compounds.Intercellular communication systems in Gram-negative bacteria are often based on chemical molecules. Adding AHLs (N-acyl homoserine lactones) which involved in QS (Quorum Sensing) signaling pathway and cAMP (cyclic AMP) which involved in the regulation of the majority of genes expressed under starvation of Gram-negative bacteria can increase the cultivation efficiency of bacterioplankton in freshwater and sea water. However, cell-to-cell communication in Gram-positive bacteria including actinobacteria usually involved in autoinducing peptides. Rpf (resuscitation promoting factor), a secreted protein in Micrococcus luteus, is a bacterial cytokine, which can stimulate the resuscitation and growth of M. Luteus, as well as several other high G+C Gram-positive organisms at picomolar concentration. Rpf cognates are widespread in many genomes and have cross-species activity.Cloned Rpfc of M. tuberculosis H37RV in pET28a(+), and expressed it in E.coli BL21(DE3) with the induction of IPTG. After Ni²⁺ affinity chromatograph, the purified rRpfC reduced the lag phase of dormant M. luteus cells when incubated in LMM (Lactate minimal media) with low density (100cells/mL) at pM level. The SN (supernatant) of M. luteus in late exponential phase was also test the biological activity by the same essay method. Samples were collected in Daihu Lake on Jinyun Mountain Beibei Chongqing. Abiotic factors were detected:temperature 15.8°C, pH 7.38, TOC 4.91mg/mL, COD_cr 27.5mg/mL. Total cell counts determined by epifluorescence after staining with PI (Propidium Iodide) were 1.29×10⁶ cells/mL. MPN (most probable numbers) was employed to count the viable cells in the samples. The cultivation efficiency was Ca. 0.2% when adding Rpf into resuscitation media. There was no difference from control. Metagenome of the environmental sample and the total DNA of cultures by MPN was extracted by modified bacteria genome extract kit. V3 to V5 region 16S rDNA were amplified by PCR. DGGE (Denaturing Gradient Gel Electrophoresis) profiles of PCR products show the bacterial diversity was improved when adding Rpf, but there are some differences between the effect of rRpfC and SN (supernatant) of M.luteus.Increasing the culturability of environmental samples mediated by Rpf could be a useful strategy to isolate as yet unculturable microorganisms and to identify new natural metabolites.