Molecular Mechanism Of Biofilm Formation In Shewanella Putrefaciens

Biofilms are described as the aggregates of microorganisms in which cells are embedded in a self-produced matrix that are adherent to each other or a surface,which impose greatly on human life.Shewanella putrefaciens CN32 is widely used in environmental remediation and microbial fuel cells.Biofilm can improve its resistance to toxic pollutants and electricity production performance,but the molecular mechanism of biofilm formation by S.putrefaciens CN32 is largely unknown.Thus,the investigation of the underlying molecular mechanism of biofilm formation by S.putrefaciens CN32 can provide the guideline for its application.Approximately 10,000 kanamycin-resistant S.putrefaciens CN32 mini-Tn5 transposon insertion mutants were screened for altered biofilm formation ability using the plate assay.These screening experiments and subsequent identification led to the selection of 73 insertion mutations,2 of which were inserted into a different locus in sputcn32₀303 which encodes a histidine kinase?HK?that had one transmembrane region.The biofilm biomass of the ?0303 mutant,sputcn32₀303 gene replacement mutant,increased significantly compared to that of the wild-type?WT?strain,indicating that the presence of Sputcn32₀303 negatively regulated biofilm formation by S.putrefaciens CN32.Sputcn32₀303 and the two proteins encodes by sputcn32₀304 and sputcn32₀305 upstream sputcn32₀303 act as a three-component regulatory system by bacterial two-hybrid experiments and pull-down experiments,which consist of HK Sputcn32₀303 and two response regulator?RR?,Sputcn32₀304 and Sputcn32_<sub>0305.Two single mutants of Sputcn32₀304 and Sputcn32₀305,A0304 and ?0305,were constructed.The biofilm biomasses of two single mutants of sputcn32₀304 and sputcn32₀305,?0304 and ?0305,were significantly increased compared to that of the WT strain,indicating that both proteins negatively regulated biofilm formation under static conditions.Besides,we constructed double mutants and a triple mutant with mutations of these three genes.The biofilm biomasses formed by all seven mutants were essentially similar and significantly higher than that of the WT strain,indicating thatthe three-component regulatory system was involved in the same signal transduction pathway.The live biofilms structures of WT??0303??0304 and ?0305 under hydrodynamic conditions monitored by confocal laser scanning microscopy?CLSM?suggested that the three-component regulatory system negatively regulates biofilm development by S.putrefaciens CN32.The intracellular c-di-GMP concentrations in the WT and ?0305 mutant were examined by LC-MS and the results suggested that RR Sputcn32₀305 did have PDE activity in vivo.The second RR Sputcn32₀304 is LuxR/GerE family transcriptional regulators and the results of qRT-PCR,EMSA,5'-RACE suggested that Sputcn32₀304 promoted sputcn32₀305 transcription directly.To mimic and prevent phosphorylation,we construct the mutants with site-directed mutagenesis of the phosphorylation residues of Sputcn32₀304 and Sputcn32₀305 protein and the biofilm analysis suggested that the negative effects of Sputcn32₀304 and Sputcn32₀305 on biofilm depend on their phosphorylation state in vivo.Further investigations confirm that Sputcn32₀303 responded to carbon source sodium lactate.Finally,Sputcn32₀303-Sputcn32₀304-Sputcn32₀305 three-component regulatory system negatively regulates the transcription of bpfA by modulating the c-di-GMP level in vivo.Changes of some environment elements are the reason of biofilm formation.Previous studies demonstrate starvation conditions can induce biofilm formation by multiple bacteria species,but the underlying regulatory mechanisms remain largly unknown.Here,we found a three-component regulatory system responds to carbon source sodium lactate and negatively regulate biofilm formation by S.putrefaciens CN32.We describe the underlying regulatory mechanisms that carbon source limited condition induces biofilm formation by S.putrefaciens CN32.This study found the underlying regulatory mechanisms that starvation conditions induced biofilm formation for the first time and served as a good example for biofilm development in bacteria.