| As the largest biological habitat on earth,the deep-sea environment contains a rich variety of microorganisms.In the deep sea,peptidoglycan is an important component of particulate organic matter(POM),and the concentration of D-amino acids,especially D-alanine(D-Ala),is very high.However,little is known about peptidoglycan turnover and D-Ala cycling in deep sea.The elucidation of these questions will shed light on the cycling of sedimentary organic matter.The low thermostability of marine thermolabile enzymes severely limits their application in biotechnology,so it's essential to develop a universal stabilizer for these thermolabile enzymes.Elastin is also an important component of marine POM,which is glycine-rich as the same as peptidoglycan.However little is known about the degradation mechanism of elastin in the ocean.Pseudoalteromonas sp.CF6-2 is a deep-sea bacterium that produces an extracellular M23 family protease pseudoalterin.In this dissertation,the bacterial competition between Ps.sp.CF6-2 and other marine bacteria was firstly examined,and the ability of Ps.sp.CF6-2 to lyse and utilize the cells of other marine bacteria for growth was demonstrated.Then the ecological role of Ps.sp.CF6-2 in deep-sea peptidoglycan turnover and the leading role of pseudoalterin in the lysis of other marine bacteria by Ps.sp.CF6-2 were studied.Next the ecological role of Ps.sp.CF6-2 in deep-sea D-Ala cycling and the D-Ala metabolism pathway of Ps.sp.CF6-2 were elucidated.In addition,a compound stabilizer with the ability to improve the thermostability of multiple thermolabile enzymes was developed.Finally,the elastin degradation mechanisms of two elastases,pseudolysin and myroilysin,were elucidated.1.Bacterial competition between Ps.sp.CF6-2 and other marine bacteriaThe battle for resources through which organisms survive and pass on genes to the next generation can often be fierce and unforgiving.Most microorganisms face a constant battle for resources,especially in oligotrophic environments,such as deep-sea sediments.Secondary metabolites,e.g.antibiotics,are used by bacteria for interspecific and intraspecific competition.All proteases in the M23 family are amidases and/or endopeptidases having peptidoglycan degradation activity,the main role of which is to be used by bacteria for defending or feeding.Firstly,the inhibitory action of Ps.sp.CF6-2 on other marine bacteria was detected.The results suggested that Ps.sp.CF6-2 could inhibit the growth of various genera of marine bacteria.Then,the interaction between Ps.sp.CF6-2 and other marine bacteria was investigated by co-culture technique which simulated the marine environment.The results demonstrated the ability of Ps.sp.CF6-2 to lyse and utilize the cells of various typical marine bacteria for growth.In addition,the non-contact interaction between Ps.sp.CF6-2 and other marine bacteria was investigated by Transwell(?)permeable supports.The results suggested that the lysis and utilization of typical marine bacteria by Ps.sp.CF6-2 was performed with the assistance of a bacteriolytic factor.Next,by testing the effect of pseudoalterin on various genera of marine gram-positive and gram-negative bacteria,we found that the lysis effect of pseudoalterin on marine bacteria was universal.Compared with gram-negative bacteria,gram-positive bacteria were more sensitive to pseudoalterin.Bacterial competition was reported to change the yield of secondary metabolites.The yield of pseudoalterin was detected when Ps.sp.CF6-2 was co-cultured with other marine bacteria.The results suggested that the cells of other marine bacteria induced the production of pseudoalterin by Ps.sp.CF6-2,and marine bacterial peptidoglycan also could induce the production of pseudoalterin.It is the first time to systematically study the competition between Ps.sp.CF6-2 and other marine bacteria,which sheds light on the deep-sea bacterial interactions.2.Ecological role of Ps.sp.CF6-2 in deep-sea peptidoglycan turnoverPeptidoglycan is a significant component of marine POM.Because of its complex structure and the existence of D-amino acids,peptidoglycan is less degradable than proteins.Currently,little is known about its turnover.Firstly,the ability of Ps.sp.CF6-2 to lyse and utilize peptidoglycan for growth was examined.When cultured in the medium with peptidoglycan as the sole carbon and nitrogen source,Ps.sp.CF6-2 degraded and utilized marine bacterial peptidoglycan for its growth.Then,we investigated the effect of peptidoglycan on the expression level of psn and the secretion of pseudoalterin by biochemical assay and RT qPCR experiment.The results suggested that both purified peptidoglycan and the peptidoglycan released from marine bacteria by bacteriophage-induced lysis induced the production of pseudoalterin by Ps.sp.CF6-2.Next,the leading role of pseudoalterin in the lysis of marine bacterial peptidoglycan by Ps.sp.CF6-2 on was examined through gene knockout and complementation experiments.By testing the lysis effect of pseudoalterin on peptidoglycan isolated from various genera of marine bacteria,we found that the lysis effect of pseudoalterin on peptidoglycan was universal,that is,pseudoalterin could degrade peptidoglycan of both gram-positive and gram-negative bacteria with high efficiency.In order to study the peptidoglycan degradation mechanism of pseudoalterin,we analyzed the cleavage sites of pseudoalterin on marine bacterial peptidoglycan.The result suggested that pseudoalterin functioned as both an amidase and an endopeptidase.In addition to the glycyl bonds in peptide bridges,the amido bond N-acylmuramoyl-Ala and peptide bonds L-Ala-D-Glu and that around lysine(Lys)were hydrolyzed by pseudoalterin.It is the first time to systematically study the function of a deep-sea M23 family protease in marine peptidoglycan turnover.Our study indicated that Ps.sp.CF6-2 and pseudoalterin may play a significant ecological role in deep-sea peptidoglycan turnover and sedimentary organic matter cycling.Except for a group of amino acids,such as L-Ala,D-Ala,D-Glu,and either Lys or diaminopimelic acid(DAP),peptidoglycan consists of two sugar derivatives,N-acetylglucosamine(NAG)and N-acetylmuramic acid(NAM).In order to find the carbohydrate hydrolase to degrade the glycan chain of peptidoglycan,the extracellular proteases profile of Ps.sp.CF6-2 was analyzed.The results suggested that N-acetyl-beta-hexosaminidase may degrade the glycan chain of peptidoglycan.However,gene knockout and heterologous expression results suggested that the enzyme had no effect on peptidoglycan lysis and utilization of Ps.sp.CF6-2,and had no peptidoglycan degradation activity.3.Ecological role of Ps.sp.CF6-2 in deep-sea D-Ala cyclingIt was reported that,in deep-sea sediments,D-amino acids accounted for up to 59%of total hydrolysable amino acids(THAA),among which the abundance of D-Ala was the highest.However little is known about the recycling of D-Ala in the ocean.Firstly,we developed a new method to analyze the amino acid enantiomers by using circular dichroism(CD).The quantitative standard curves of alanine enantiomers or glutamate enantiomers were established,which showed a perfect linear relationship between amino acids concentration and CD spectra data.Compared with widely-used methods such as capillary electrophoresis(CE),high performance liquid chromatography(HPLC)and gas chromatography(GC),our method has the following advantages:no derivatization,easy operation,good reproducibility,qualitative and quantitative determination.It is a new attempt to use CD in amino acid enantiomers determination,which sets up a new technique for chiral recognition.Secondly,we investigated the utilization of D-Ala and D-Glu by Ps.sp.CF6-2.The results showed that Ps.sp.CF6-2 could utilize D-Ala or D-Glu as the sole nitrogen source,which indicated that Ps.sp.CF6-2 is a deep-sea bacterium with the ability for D-amino acid mineralization.To determine the mechanism how Ps.sp.CF6-2 mineralizes and utilizes D-Ala,the whole transcriptomes of Ps.sp.CF6-2 grown in L-Ala and D-Ala were sequenced and compared.The results showed that genes related to D-Ala metabolism were up-regulated when Ps.sp.CF6-2 cultured in D-Ala,such as racA1 and racA2 that encode D-Ala racemase,traA that encodes D-Ala aminotransferase and ligA that encodes D-Ala--D-Ala ligase.Then the roles of these genes in the D-Ala utilization of Ps.sp.CF6-2 were examined through gene knockout and complementation experiments.The results demonstrated that genes traA and racA1 played key roles in the D-Ala utilization of Ps.sp.CF6-2.Our study elucidated the D-Ala metabolism pathway of Ps.sp.CF6-2.Analysis of the genomes of 70 bacteria isolated from deep-sea sediments and hydrothermal vents,which are in GenBankTM database,indicates that most of these bacteria have one or several M23 family proteases and the key enzymes involved in D-Ala metabolism we elucidated.This finding suggests that the M23 family proteases,D-Ala racemase,D-Ala aminotransferase and D-Ala--D-Ala ligase may be popular in deep-sea sediments,and therefore,play important roles in sedimentary POM releasing and D-amino acids recycling.4.Thermostability improvement of pseudoalterinPseudoalterin is the most abundant protease secreted by the marine sedimental bacterium Ps.sp.CF6-2 and is a novel cold-adapted metalloprotease of the M23 family.Proteases of the M23 family have high activity towards peptidoglycan and elastin,suggesting their biomedical and biotechnological potentials.Pseudoalterin has the ability to lyse bacterial cell walls,and therefore has promising therapeutic values for the treatment of antibiotic-resistant infections in humans.However,it is a cold-adapted enzyme with low thermostability.The half-life of pseudoalterin activity was only 14.13 min at 35?,and the enzyme lost almost 60%of its original activity after 24 days at 4?.By vacuum freezing drying technique,we developed a method to transform liquid pseudoalterin into dry powder.Compared with liquid enzyme,the thermostability of dry powder was improved.At 4?,dry powder of pseudoalterin retained 97%of its original activity after 20 days and 86%after 60 days.What's more,dry powder is beneficial for the transportation and storage of pseudoalterin.Through single factor experiments and orthogonal tests,we developed a compound stabilizer by using medically safe sugars and polyols.The stabilizer had a significant protective effect for pseudoalterin against thermal denaturation.In the presence of the compound stabilizer,pseudoalterin at 37? retained 80%of its original activity after three days and 50%after five days.At 4?,the activity of pseudoalterin preserved in the presence of compound stabilizer remained quite stable after 360 days.The compound stabilizer lays a solid foundation for the applications of pseudoalterin.In addition,this compound stabilizer also improved the thermostability of multiple other enzymes,indicating that it may be used as a universal stabilizer for thermolabile enzymes.5.Mechanistic insights into elastin degradation by pseudolysin and myroilysinIn recent years,significant progress has been made on the assembly and structure of elastin.Pseudomonas aeruginosa is an opportunistic human pathogen that can cause eye infections,pulmonary infections,bacteremic infections and burn infections.Pseudolysin is the most abundant protease secreted by P.aeruginosa,and is considered as the predominant extracellular virulence factor of P.aeruginosa.Elastin is an important constituent of sedimentary particulate organic nitrogen(PON).However,there are relatively few studies on marine elastases.Myroides profundi D25 is a protease-producing bacterium that we isolated from the deep-sea sediment near the southern Okinawa Trough.Strain D25 secretes a novel astacin-like metalloprotease,myroilysin,which is the most abundant protease secreted by this strain.In addition to its high elastin-degrading activity,myroilysin also has strong collagen-swelling ability and plays a synergistic role with collagenase in collagen hydrolysis.Our study sheds lights on the pathogenesis of P.aeruginosa infection and the biotechnological applications of myroilysin.Firstly,we investigated the elastin binding mechanism of pseudolysin by biochemical assay,and found that hydrophobic and electrostatic interactions existed between pseudolysin and elastin.Moreover,site-directed mutational results indicated that the hydrophobic residues in the S1' sub-site,Val137,Leu197,Leu132,and Phe129,and Tyr114 in the S1 sub-site played key roles in the binding of pseudolysin to insoluble elastin.These aliphatic and aromatic residues in the S1-S1' sub-sites of pseudolysin may serve as the targets for designing specific drugs for blocking the elastin binding of pseudolysin in P.aeruginosa infection.In addition,we examined the elastin binding and degradation mechanism of myroilysin.Myroilysin bound hydrophobically when mixed with insoluble bovine elastin,suggesting that this elastase may interact with the hydrophobic domains of elastin.Consistent with this,analysis of the cleavage pattern of myroilysin on bovine elastin and recombinant tropoelastin revealed that myroilysin preferentially cleaved peptide bonds with hydrophobic residues at the P1 and/or P1' positions.Scanning electron microscopy(SEM)of cross-linked recombinant tropoelastin degraded by myroilysin suggested preferential damages of spherules over cross-links,as expected for a hydrophobic preference.Our results are helpful for developing biotechnological applications for myroilysin.In conclusion,this study sheds light on the bacterial competition,the turnover of microbial peptidoglycan and the recycling of D-alanine in the oceanic biosphere driven by sediment bacterium Ps.sp.CF6-2 and its extracellular enzyme pseudoalterin,leading to a better understanding for the cycling of sedimentary organic matter.In addition,elastin is also an important component of marine POM.A compound stabilizer with the ability to improve the thermostability of elastase pseudoalterin was developed,and the elastin degradation mechanisms of two elastases,pseudolysin and myroilysin,were elucidated. |
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