Marine Bacterial Metabolism Of D-Alanine And Diaminopimelic Acid,Key Components Of Bacterial Cell Wall Peptidoglycan

The ocean covers more than 70%of the earth's surface,which contains microorganisms abundant in both quantity and variety.Bacterial cell wall peptidoglycan is an important component of particulate organic matter(POM)in the ocean.D-alanine(D-Ala)is a key component of peptidoglycan,and diaminopimelic acid(DAP)is a characteristic component of Gram-negative bacterial peptidoglycan.In the marine environment,the mechanism by which microorganisms use D-Ala and DAP,and the types of bacteria that can utilize DAP,are currently unclear.Pseudoalteromonas sp.CF6-2 is a strain isolated from deep-sea sediments that can degrade Gram-positive peptidoglycan and grow with D-Ala as the sole nitrogen source.In this thesis,three key enzymes in the metabolic pathways of D-Ala of CF6-2 were heterologously expressed and purified,and their in vitro function and characters were studied.DAP is a characteristic component of Gram-negative bacterial peptidoglycan.In this thesis,the diversity of bacteria that can utilize DAP as the sole carbon and nitrogen source in seawater samples collected from multiple western Pacific sites with varied sampling depths was analyzed,and 45 strains capable of utilizing DAP as the sole carbon and nitrogen source were isolated.This thesis also analyzed the metabolic pathway of DAP of two isolated strains.1.In vitro functional verification and characterization of key enzymes in the D-Ala metabolic pathways of deep-sea bacterium Pseudoalteromonas sp.CF6-2.Previous studies have shown that Ps.sp.CF6-2 secretes an elastase,pseudoalterin,to cleave the peptide bond at both ends of D-Ala in Gram-positive peptidoglycan,releasing the D-Ala molecule,and can grow with D-Ala as the sole nitrogen source.Genome and transcriptome analyses suggest that D-Ala ligase 0208,Ala racemase 3059 and D-Ala aminotransferase 1 756 are key enzymes in the metabolic pathways of D-Ala in Ps.sp.CF6-2.In this thesis,the in vitro function of these three enzymes were verified,and their characters were studiedD-Ala is generally toxic to bacteria.We determined the growth of Ps.sp.CF6-2 with different concentrations of D-Ala as the sole nitrogen source.The results showed that,although Ps.sp.CF6-2 could grow with D-Ala as the sole nitrogen source,high concentrations of D-Ala inhibited the growth of Ps.sp.CF6-2,indicating that Ps.sp.CF6-2 may be able to tolerate the toxicity of low concentrations of D-Ala by converting it to other substances.Three key enzymes in the D-Ala metabolic pathways of Ps.sp.CF6-2,D-Ala ligase 0208,Ala racemase 3059 and D-Ala aminotransferase 1756,were heterologously expressed and purified.The function of these three enzymes was verified by measuring their activity in vitro.The results showed that D-Ala ligase 0208 could catalyze the linkage of D-Ala to produce D-Ala-D-Ala dipeptide,Ala racemase 3059 could catalyze the conversion of D-Ala and L-Ala to each other,and D-Ala aminotransferase 1756 could catalyze the deamination of D-Ala to produce pyruvic acid.The characters of these enzymes,including their Michaelis-Menten curves,were also studied.2.Diversity analysis and isolation of bacterial strains in western Pacific water samples that can utilize diaminopimelic acid.Bacteria in the 16 seawater samples collected from different depths in Western Pacific sites A1,A3,A4,A10,and B3 were enriched with the medium containing DAP as the sole carbon and nitrogen source.After enrichment,the diversity of bacteria in the western Pacific water samples that can utilize DAP was analyzed.Bacteria that can utilize DAP in the western Pacific surface and shallow seawater samples are mainly in the genera of Thalassospira,Erythrobacter,and Halomonas,and those in the deep seawater samples are mainly in the genera of Alteromonas,Pseudoalteromonas,Microbacterium,Thalassospira,and Nitratireductor.We then isolated 45 strains that can utilize DAP from the above-mentioned seawater samples using DAP screening plates.3.Analysis of the metabolic pathways of diaminopimelic acid in western Pacific bacteria strains.To analyze the DAP metabolic mechanism of marine bacteria,we selected two strains,one Gram-positive and the other Gram-negative,that can utilize DAP as the sole carbon and nitrogen source and grow relatively fast,and sequenced their genomes By analyzing the gene annotations in their genomes and referring to the relevant enzymology databases,we speculated that they utilize DAP primarily through two pathways:using diaminopimelate decarboxylase to convert DAP to lysine and then into the citric acid cycle;or using UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase and UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase to link DAP to a short peptide linked to an acetyl base to form UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine,which is an important raw material for the DAP-type cell wall peptidoglycan of Gram-negative bacteria,and can be directly used by the Gram-negative bacteria for the biosynthesis of peptidoglycan of its own cell wallThe results in this thesis preliminarily revealed the mechanisms of marine bacteria to metabolize D-Ala and DAP,which have important significance in clarifying the mechanisms of the biogeochemical recycling of D-Ala and DAP driven by marine bacteria.