| Deep sea is a special ecological environment with dark,cold,hyperhaline and high-pressure all year round.There are a rich variety of microorganisms in the deep-sea sediments.They can secrete various extracellular proteases to degrade the organic nitrogen in the environment for their own growth and reproduction,and they also play an important role in the marine organic nitrogen cycling.However little is known about that how deep-sea microorganisms sense the organic nitrogen in the environment and then initiate the expression of extracellular proteases.Moreover,how do the decomposed amino acids and oligopeptides be transported to cells is still unknown.Pseudoalteromonas sp.SM9913,isolated from deep-sea sediments,is a deep-sea bacterium with high extracellular protease production.SM9913 can secrete a variety of extracellular proteases in the medium with casein as the sole nitrogen source and the serine protease MCP-01 is the most predominant.Preliminary studies have shown that the di-/tripeptide transporter Dtp of SM9913 might be related to the induction of MCP-01.In this study,the roles of Dtp in the transport of peptides and the induction of extracellular protease were preliminarily revealed by means of physiology,biochemistry,transcriptomics and molecular genetics.1.The construction of the complementary strains and point/truncated mutants based on the deletion mutant strain ?dtp.In order to better understand the role of the di/tripeptide transporter Dtp in the peptide transport and the induction of extracellular proteases,we constructed the dtp-complementary strain dtp-pEV-?dtp and the empty vector-complementary strain pEV-?dtp on the basis of the previously constructed deletion mutant strain Adtp.The results of Western Blot showed that the expressions of Dtp were observed on cell membranes of SM9913 and dtp-pEV-?dtp,while not on the ?dtp and pEV-?dtp,The results further confirmed that Dtp could express and play a role on the cell membrane of SM9913.To further elucidate the molecular mechanism of Dtp on transporting peptides,six key amino acids that might bind to protons or substrates of Dtp were predicted,based on the crystal structure of Dtp homologues.The site-directed mutations of these key amino acid residues were constructed,and then seven point mutants were obtained by complementing the point-mutated gene of dtp into ?dtp.In addition,in order to study the structural basis and molecular mechanism of Dtp involved in sensing peptides and transferring signal,regions that might transmit signals into the cell in Dtp were predicted by structural analysis,and deletion mutations of these key regions were performed.Two truncated mutants were obtained by complementing the deletion-mutated genes of dtp into ?dtp.The construction of the complementary strains and mutants lays the foundation for further study on the function and mechanism of Dtp as a transceptor in the transport of peptide and the induction of extracellular protease.2.Study on the transport function of the di-/tripeptide transporter Dtp in SM9913.To study whether the di-/tripeptide transporter Dtp has the ability to transport dipeptides or tripeptides,the wild-type strain,peptide-transport-system deletion mutants,and complementary strain were cultured in the medium with dipeptides or tripeptides as the sole nitrogen source and their growth were analyzed.The results showed that SM9913 could grow in dipeptides and tripeptides medium,and the growth of ?dtp was significantly inhibited.On the contrary,the medium had no effect on the growth of ?dppA.As expected,the growth of dtp-pEV-?dtp was restored.These results indicated that Dtp had the ability to transport dipeptides and tripeptides.In order to observe simply the transportation ability of Dtp for tripeptides,the transportability of SM9913 for different types of tripeptides was studied,and two tripeptides that had higher transport activity were selected for FITC-fluorescent labeling.After incubating with FITC-labeled tripeptides,fluorescence could be observed at the intracellular region of SM9913 using fluorescence microscopy,which demonstrated that tripeptides could be transported into the intracellular by Dtp.To elucidate the molecular mechanism of the Dtp for peptides transport,seven point mutants were cultured with the tripeptides as the sole nitrogen source.The results showed that the growth of E300A-pEV-?dtp was most significantly inhibited,and it was known that Glu300 was the key amino acids that might combine with substrate,demonstrating that the ability of Dtp for tripeptides transport might closely related to its ability to bind substrates.These results confirmed the di-/tripeptide transporter Dtp had the ability to transport dipeptides and tripeptides.We speculated that the Glu300 might be the key amino acid that was related to bind substrate in Dtp and it could influence the transport function of Dtp.3.Transcriptomic analysis on the role of di-/tripeptide transporter Dtp from Ps.sp.SM9913 in the induction of extracellular protease.To study the relationship between Dtp and the induction of extracellular protease in SM9913,we cultured SM9913 and ?dtp in casein medium and performed transcriptome sequencing and bioinformatics analysis.Under casein-induced conditions,20 genes of extracellular protease including MCP-01 in SM9913 were up-regulated,and the expression of mcp01 was the highest.dtp was also up-regulated.In the mutant ?dtp,the expression of 12 genes of extracellular protease including MCP-01 were significantly decreased compared with SM9913.Among them,the expression of mcp01 in ?dtp was much lower than that in SM9913,and dtp was not expressed.The results were consistent with the results of RT-qPCR,both demonstrating that the deletion of dtp could affect the expression of extracellular proteases induced by casein,and Dtp might have the function as receptors for sensing extracellular signals during the induction of extracellular protease of SM9913.Taken together,our findings preliminarily revealed that the di-/tripeptide transporter Dtp might play a role as a transceptor in SM9913,and we proposed the possible functional mechanism of Dtp as follows.In the culture medium with casein as the sole nitrogen source,the basally expressed extracellular proteases of SM9913 degrade the casein in the environment into oligopeptides which initiate the upregulation of dtp for survival of SM9913.Then Dtp transports the oligopeptide products into the cell to provide energy.In addition,Dtp can also sense these oligopeptides and transmit the signals of extracellular nutrients into the cell.Under a series of signal transduction and the function of multiple transcriptional regulatory factors in the cell,the expression and secretion of extracellular proteases could be initiated and then promoted the extracellular proteases to further degrade and use the extracellular nutrients.In the meantime,the up-regulation of a series of genes related transporters including Dtp was initiated to promote the absorption and utilization of substrates.Therefore,Dtp might be a transceptor that retains the transport function in SM9913. |
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