Study On Marine Bacteria Utilizing Gram-negative Bacterial Cell Wall Peptidoglycan And Their Extracellular Enzymes

The ocean occupies more than 70%of the earth's surface area,which contains a huge number and variety of microbial resources.Due to the unique structure,peptidoglycan of Gram-negative bacteria is difficult to be degraded,becoming a very important component of particulate organic matter(POM)in the ocean.The best studied predators of bacteria are Bdellovibrio and Bdellovibrio-like bacteria(BALOs),which prey on a wide range of Gram-negative bacteria.However,little is known about other predators of Gram-negative bacteria in the ocean.The mechanism of the degradation,absorption and utilization of peptidoglycan of marine Gram-negative bacteria and the extracellular enzymes involved in the degradation still remains unclear.It is of great significance to reveal the role of microorganisms in carbon and nitrogen cycle of the ocean.And it is important to study on the peptidoglycan degrading bacteria and enzymes for understanding the carbon and nitrogen cycle in the ocean.In this study,three peptidoglycan degrading strains of Vibrio rumoiensis 3A-4,Pseudoalteromona.s agarivarons 10-9 and Pseudoalteromonas hodoensis 1-1 were screened from the West Pacific and offshore seawater and the competitive relationship between these three strains and other typical marine Gram-negative bacteria were studied.It was verified that these strains are capable of lysing and utilizing typical marine bacteria.Moreover,the diversity of extracellular enzymes of marine bacteria V.rumoiensis 3A-4 was analyzed on a genome-wide level,and the extracellular enzymes that might be related to the degradation of Gram-negative peptidoglycan were predicted.1.Screening of strains using gram-negative bacterial cell wall peptidoglycan from seawater samplesIn order to screen-strains capable of degrading Gram-negative bacterial peptidoglycans in the ocean,we used 2216E and Gram-negative peptidoglycan culture medium to enrichment culture seawater samples from different depths at multiple sites in the western Pacific and offshore(Weihai Rongcheng,Tianheng Island,and Pacific Ocean),respectively.The diversity of bacteria in the enriched culture medium was analyzed,and the degradation strains were screened by the plate screening method using Gram-negative bacterial peptidoglycan as the sole carbon and nitrogen source.Three strains utilizing Gram-negative bacterial peptidoglycans were obtained,namely V rumoiensis 3A-4?P.agarivarons 10-9 and P.hodoensis 1-1.2.Competitive relationships between V.rumoiensis 3A-4?P.agarivarons 10-9,P.hodoensis 1-1 and typical marine bacteriaThere is intensively competition among microbial communities for competing and protecting the growing resources and space.In this study,we used Pseudoalteromonas.sp.CF6-2 as the testing strain to study the lysis effect of V.rumoiensis 3A-4?P.agarivarons 10-9 and P.hodoensis 1-1 on marine bacteria with plate bacteriostatic experiment and a permeability culture device in a non-contact mode.It was found that V.rumoiensis 3A-4 was capable of producing diffusible lysis factor to utilize P.sp.CF6-2.Moreover,we studied the growth inhibition effects of different genera of marine bacteria on V.rumoiensis 3A-4?P.agarivarons 10-9 and P.hodoensis 1-1,by using a semi-permeable culture device in a non-contact mode.It was found that inhibition effects of V.rumoiensis 3A-4?P.agarivarons 10-9 and P.hodoensis 1-1 were genera dependent.3.Analysis of bacterial extracellular enzymes involved in Gram-negative peptidoglycan degradation by genome sequencingTo analyze the extracellular enzymes involved in the degradation of Gram-negative peptidoglycan,we sequenced the whole genome of V rumoiensis 3 A-4.It was found that there are 52 genes encoding extracellular enzymes in the genome of V.rumoiensis 3A-4,19 of which are peptidase genes with signal peptides.Through gene function analysis,13 extracellular enzymes were predicted to be involved in the degradation of Gram-negative peptidoglycan,including 8 proteases,2 amidases and 3 glycosidases.These enzymes may play important roles in the degradation of the cell wall peptidoglycan of marine bacteria and involve in carbon-nitrogen cycle of the ocean.These results laid a foundation for better understanding the important role of marine bacteria in the degradation cycle of marine organic carbon and nitrogen.