Mechanistic Insight Into The Expression Induction,Secretion,Catalysis And Ecological Role Of The Extracellular Protease Pseudoalterin From A Deep-sea Sedimental Bacterium

The deep-sea environment contains huge and various microbial resources.Microorganisms are the most important organisms in deep-sea ecosystem.There are complicated interactions in deep-sea microorganisms,which,however,have been little known.Most organic matters in deep sea are particulate organic matters(POM)with high molecular weight.Deep-sea microorganisms degrade the POM by secreting enzymes and utilize the degradation products to adapt to the deep-sea environment.Peptidoglycan from marine bacteria and collagen from marine animals are important components of deep-sea POM,which are degraded by microbial extracellular enzymes and utilized by deep-sea microorganisms,thereby entering deep-sea carbon and nitrogen cycling.However,our knowledge about the microorganisms and the enzymes involved in the degradation of deep-sea POM including peptidoglycan and collagen and their degradation mechanisms is still quite limited.Pseudoalteromonas is a group of heterotrophic bacteria widely distributed in the marine environment.Pseudoalteromonas sp.CF6-2 is a protease-secreting bacteria isolated from the deep-sea sediment from the South China Sea.Previous study showed that the extracellular protease Pseudoalterin secreted by CF6-2 is a M23 metalloprotease that can degrade elastin and peptidoglycan from Gram positive bacteria.On this basis,in this dissertation,a genetic manipulation system of CF6-2 and a cold-adapted expression system for Pseudoalterin expression were first constructed.Then,the mechanisms for the expression induction,maturation,secretion and catalysis of Pseudoalterin of CF6-2 were studied,the ecological role of strain CF6-2 to prey marine Gram positive bacteria via Pseudoalterin was revealed,and the small and pilot fermentation technologies of CF6-2 for Pseudoalterin production were developed.In addition,the characters of a new S8 serine protease P57 secreted by Photobacterium sp.A5-7 isolated from Jiaozhou Bay sediment and the function of the PA domain in P57 were studied.Results in this dissertation are as follows.1.the diversity of extracellular proteases in marine bacteria at the genome levelIn order to investigate the diversity of extracellular proteases in marine bacteria at the genome level,the genomes of P.sp.CF6-2 from deep-sea sediment and Flavobacterium arcticum SM1502T from Arctic surface seawater were sequenced.Based on gene function annotation,the diversity of extracellular proteases,and other extracellular enzymes of these two marine bacteria was analyzed at the genome level.CF6-2 encodes 50 peptidases with signal peptides.These peptidases include 28 serine peptidases distributing in 9 serine peptidase families and 22 metallopeptidases distributing in 15 metallopeptidase families.SM1502T encodes 29 peptidases with signal peptides.These peptidases include 14 serine peptidases distributing in 7 serine peptidase families,11 metallopeptidases distributing in 5 metallopeptidase families and 4 cysteine peptidases distributing in 4 cysteine peptidase families.These results indicated that both CF6-2 and SM1502T contains many extracellular protease genes and therefore,likely play an important role in the degradation and recycling of marine organic nitrogen.2.The induction mechanism for the expression of extracellular protease Pseudoalterin in P.sp.CF6-2The production of most bacterial extracellular proteases is induced by outside nutrients.However,till now,no signal molecules to induce the production of bacterial extracellular protease have been identified.We identified the signal molecules to induce the expression of extracellular Pseudoalterin of CF6-2.Because Pseudoalterin can efficiently degrade the peptide chain of peptidoglycan from Gram positive bacteria,we supposed that some components of peptidoglycan may have inducing effect on the expression of Pseudoalterin.Therefore,we tried to identify the inducing signal molecules for Pseudoalterin expression from the components of peptidoglycan.We first analyzed the inducing effect of the peptidoglycan from Staphylococcus warneri MCCC0423,a typical marine Gram positive bacterium,on the transcription of Pseudoalterin.Then,we synthesized 19 oligopeptides according to the sequence of the peptide chain of peptidoglycan,and test the inducing effect of each peptide and each amino acid in the peptide chain on the transcription of Pseudoalterin.The result showed that Glycine and Glycine oligopeptides had inducing effect,and these molecules are the inducing signal molecules for Pseudoalterin expression.The effective concentrations of Glycine to induce Pseudoalterin expression are 0.25 mM-20 mM.This is for the first time to identify the inducing signal molecules for microbial extracellular proteases and determine its effective concentrations.In addition,the transcriptional initiation site of pseudoalterin was identified,which laid a foundation for further elucidation of the intracellular regulation mechanism of CF6-2 on Pseudoalterin synthesis.3.The maturation and secretion of extracellular protease Pseudoalterin in P.sp.CF6-2Previous study has shown that the M23 protease Pseudoalterin cannot self-mature,and other proteins are needed to cleave the propeptide during Pseudoalterin maturation.However,it is unclear which proteins are involved in the cleavage of the propeptide during Pseudoalterin maturation.In order to identify these involved proteins and to clarify the maturation mechanism of Pseudoalterin,we first constructed the genetic manipulation system of CF6-2 and predicted the possible proteins involved in the cleavage of Pseudoalterin propeptide by referring to the maturation process of other M23 family proteases.Then,the genes of these proteins were knocked out using the constructed genetic manipulation system of strain CF6-2,and the extracellular enzyme activity of the mutants was measured.Finally,three proteases involved in Pseudoalterin maturation,Lysyl,Serine 1 and Serine5,were determined.The results of signal peptide prediction indicate that these three proteases are located in the periplasmic space,and the mature form of Pseudoalterin was found in the whole cell supernatant by Western blot,which suggests that the Pseudoalterin propeptide is most likely cleaved in the periplasmic space of CF6-2 cell.By analyzing the genome of the strain CF6-2,it was found that there is a complete Type ? secretion system(T2SS)in CF6-2.When the key gene of T2SS,gspE,was deleted,extracellular Pseudoalterin of the mutant strain was almost undetectable.When the gene gspE was complemented into the mutant,the secretion of Pseudoalterin was restored.Therefore,it can be sure that Pseudoalterin is secreted to extracellular via T2SS.4.The mechanism of Pseudoalterin to bind and catalyze peptidoglycan degradationPrevious study showed that Pseudoalterin has two domains.The catalytic domain contains a Zn ion and can hydrolyze the peptide chains of peptidoglycan.The C-terminal binding domain is closely arranged with the catalytic domain in a T-form and can bind to the carbohydrate chains of peptidoglycan.Although it has been shown that Pseudoalterin can effectively degrade peptidoglycan,the binding and catalytic mechanisms of Pseudoalterin for peptidoglycan degradation still remain unclear.To probe these mechanisms,the peptides AeKaA and AGGGGG from the peptide chains of peptidoglycan were molecular co-simulated with the catalytic cavity of Pseudoalterin by the molecular docking method,and the key amino acid residues involved in the interaction of Pseudoalterin and peptidoglycan in the models were predicted in combination with homologous sequence analysis.Then,we constructed a marine cold-adapted Pseudoalteromonas expression system to express active Pseudoalterin.With this expression system,the point mutants on the predicted key residues of Pseudoalterin and a C-terminal binding domain deleted mutant were expressed and purified.By measuring the degradation activity and adsorption capacity of Pseudoalterin mutants on peptidoglycan,the molecular mechanism of Pseudoalterin to bind and degrade bacterial peptidoglycan was elucidated,which represents the first revealing of the degradation mechanism of bacterial peptidoglycan by a deep-sea bacterial protease.5.The ecological role of P.sp.CF6-2 and its extracellular protease PseudoalterinThe M23 proteases secreted by bacteria can degrade bacterial cell wall peptidoglycans,providing advantages for bacteria to obtain food,defend and compete with other bacteria.To clarify the interaction of CF6-2 with marine Gram positive bacteria,we co-cultured CF6-2 with some marine Gram positive bacteria on plates,and found that CF6-2 was able to lyse a variety of marine Gram-positive bacteria and grow with the nutrients from the lyzed bacteria.This indicates that CF6-2 probably has the ability to prey on marine Gram positive bacteria.Then,with Staphylococcus warneri MCCC1A00423 as a material,the mechanism of CF6-2 to lyse marine Gram positive bacteria by secreting Pseudoalterin to degrade the bacterial peptidoglycan and to grow and multiply with nutrients from Gram positive bacteria lysis was studied.Based on our results,a model for CF6-2 to prey marine Gram-positive bacteria was proposed.In addition,through bioinformatics analysis,it was found that many marine bacteria,especially Gram negative bacteria,contain Pseudoalterin-like sequences.Therefore,it is speculated that the phenomenon that marine bacteria prey on Gram-positive bacteria by extracellular M23 proteases may be universal in the marine ecological environment.Therefore,we reveal a new prey-predation interaction between marine bacteria.6.Small and pilot fermentation of P.sp.CF6-2 for Pseudoalterin productionThe M23 family proteases can degrade peptidoglycan and elastin,and thus has application potential in biomedical and biotechnology.In order to reduce the fermentation cost and increase the yield of Pseudoalterin,the composition of the fermentation medium and the fermentation culture conditions were optimized by a single factor experiment.When 0.5%sucrose was added to the medium as a carbon source,the amount of bovine artery powder can be reduced from 1.2%to 0.6%,and the yield of Pseudoalterin was increased to 161%(161.2 ± 3.1 U/mL).Based on the optimized medium,the stirring speed and the aeration rate for CF6-2 were optimized in 5 L and 50 L fermentors,and thus,the small and pilot fermentation technologies of CF6-2 for Pseudoalterin production were developed.The yields of Pseudoalterin in the small and pilot fermentation were 155.6± 10.5 U/mL and 103.5 ± 8.6 U/mL,respectively.This result lays a solid foundation for the industrial production and the application in biomedicine and biotechnology of Pseudoalterin.7.The properties of the new S8 family serine protease P57 and the function of the PA domainIn addition to the study on the marine bacterial protease Pseudoalterin,we also studied the the properties of a new S8 family serine protease P57 secreted by Photobacterium sp.A5-7 and the function of the PA domain in P57.Photobacterium sp.A5-7 is a protease-secreting bacterium isolated from Jiaozhou Bay sediments.The extracellular protease P57 from A5-7 was purified and characterized.P57 is a new subtilisin member of the S8 family.P57 has activities towards casein,gelatin and collagen.Mature P57 has a catalytic domain and a PA domain.Analysis of the interaction of the fusion protein PA-EGFP with collagen showed that the PA domain had collagen-binding ability.In addition,the binding of the P57 protein the activity of which was inhibited by Fe3+ also could bind collagen.Site-directed mutagenesis analysis indicated that Phe349 and Tyr432 are the key amino acid residues in the PA domain for P57 binding to collagen.These results indicate that protease P57 can bind the collagen substrate through the PA domain.Our results firstly provide direct evidence for the insoluble substrate-binding ability of the PA domain in a S8 subtilisin protease,which is important to better understand of the function of the bacterial extracellular proteases from marine sediments and the function of the PA domain in subtilisins.In summary,this dissertation studied the deep-sea bacteria CF6-2 extracellular protease Pseudoalterin for its degradation of marine peptidoglycan and its ecological function of marine organism competition.At the same time,the expression induction,maturation,secretion and catalysis mechanisms of Pseudoalterin were also studied.The results are helpful to better understand the role of extracellular enzymes in the deep sea material cycling and microbial interaction.Pseudoalterin,as a protease capable of degrading peptidoglycan,has a good potential in biomedicine and biotechnology.Our results on its small and pilot fermentation for Pseudoalterin production will lay a solid foundation for its large-scale industrial production.In this dissertation,a genetic manipulation system of strain CF6-2 and a cold-adapted expression system were established,which is of great significance for the study of other cold-adapted bacteria and proteins.In addition,our study on the enzymatic properties and the function of the PA domain of protease P57 has significance for better understanding the functions of bacterial extracellular proteases from marine sediments and the PA domain in subtilisins and for elucidating the degradation of particulate organic nitrogen in marine sediments.