Study On The Structure And Function Of Wall-membrane Anchoring Protein Lpp In Gram-negative Bacteria

The periplasmic space is a characteristic cellular structure of Gram-negative bacteria.Unlike Gram-positive bacteria,the cell wall of Gram-negative bacteria is surrounded by an outer membrane structure in addition to the peptidoglycan layer.The space between the outer membrane and the inner membrane is the periplasmic space of gram-negative bacteria.As one of the most basic cellular structures unique to gram-negative bacteria,a stable periplasmic space is extremely important for the life activities of gram-negative bacteria.In order to maintain the structure of periplasmic space,the most important thing is to maintain the stability of the outer membrane structure.A variety of proteins in Escherichia coli are involved in maintaining the stability of the outer membrane structure,such as Lpp,Pal,OmpA,etc.The most important of them,and the one that plays the most important function,is Lpp.Lpp is a lipoprotein whose N-terminal three fatty acid molecules can be inserted into the outer membrane of E.coli,and whose C-terminal can be anchored to the cell wall peptidoglycan by means of covalent bonding.Lpp stabilizes the outer membrane and maintains its stability by simultaneously anchoring the peptidoglycan and attaching the outer membrane.Lpp is also the only protein found in E.coli that can be covalently linked to the peptidoglycan in the cell wall.LPP is widely found in multiple bacterial groups,such as y-Proteobacteria,Enterobacteria,Vibrio,Pseudomonas,and so on.Despite the intensive research on Lpp over the past 50 years,many key scientific questions remain unanswered.One of the most important problems is that researchers have not been able to effectively observe LPP through a variety of microscopic means,which also poses an important limitation to the further study of Lpp.This master's research project successfully realized direct observation of E.coli Lpp by using atomic force microscopy.Combined with a series of techniques including molecular,biochemical and microscopic observation,the physiological function of E.coli Lpp in cells,the effect of E.coli L,D transpeptidase genes on the protein anchoring of LPP,and the diversity of Lpp in other strains were studied in depth,which provided an important basis for the subsequent scientific research on the physiological function of Lpp.The specific research results of this paper are as follows:1.Observation and identification of E.coli cell wall binding protein Lpp.The cell walls of E.coll were observed using high resolution atomic force microscopy(AFM).Under the AFM,the cell wall surface is covered with extremely dense granular structures.After enzymatic hydrolysis experiment and mass spectrometry analysis of the enzymatic hydrolysates,it was found that the peptide obtained after enzymatic hydrolysis came from the Lpp protein of E.coli.We then knocked out the Lpp gene in E.coli,and the granular material on the cell wall of the knockout strain completely disappeared.Finally.lpp gene complement was performed on the knockout strains.and the high-density granular structures were reproduced on the cell wall.Through a series of experiments,it was finally confirmed that the particles observed on the surface of the cell wall of E.coli by AFM were Lpp proteins.2.Physiological functions of cell wall binding protein Lpp of E.coli.To explore the physiological functions of Lpp proteins on cells.we explored the WT/?Lpp/cpLpp strains in a series of physiological function.When the Lpp gene was deleted.the morphology of the bacteria changed and the vesicle structure appeared on the cell surface.There was no significant difference between the growth curve of E.coli?Lpp under normal growth conditions and that of the wild type,indicating that the loss of Lpp protein in E.coli did not affect its normal survival.And then we determine the E.coli WT/?Lpp/cpLpp strains on the osmotic pressure sensitivity,EDTA sensitivity and stress sensitivity,the results show that when the Lpp gene deletion.strain osmotic pressure sensitivity,EDTA sensitivity and stress sensitivity increased dramatically.Loss of the Lpp gene leads to impaired cell wall function,decreased periplasmic spatial stability.and reduced ability to maintain mechanical properties and resist environmental stress.3.Effect of L,D transpeptidase gene of E.coli on Lpp protein anchoring.In Escherichia coli,LdtA,LdtB and LdtC transpeptidases are involved in the process of attaching Lpp to the peptidoglycan layer of the cell wall.We performed single/double/triple knockout of L,D transpeptidase gene ldtA/ldtB/ldtC in Escherichia coli strains,and extracted cell walls for AFM observation and analysis to explore the effect of L.D transpeptidase LdtA/LdtB/LdtC on Lpp protein anchoring.In logarithmic growth phase.LdtB plays a major role in the connection of Lpp to the peptidoglycan layer of cell wall,while LdtA and LdtC play a secondary role.In the stable phase,LdtA and LdtB played a major role in the connection of Lpp to the peptidoglycan layer of cell wall,while LdtC played a secondary role.Among the three Ldts,LdtC plays a minor role in the connection of Lpp.LdtA mainly functions in the stable phase,while LdtB plays an important role in both the logarithmic phase and the stable phase.4.Diversity of cell wall binding protein Lpp in Gram-negative bacteria in other strains.By comparative analysis of Lpp from Gram-negative bacteria,it was found that Lpp was widely present in multiple bacterial groups of Gram-invisible bacteria,such as Enterobacteria,Alternative Monomonas,Vibrio,Pseudomonas,etc.The sequence of amino acid residues of Lpp protein from different bacterial species was conserved in structure.Through phylogenetic tree analysis,we found that Lpp from different orders generally clustered in the same cluster.This indicated that the conserved degree of Lpp sequence was related to the taxon of the strain from which it originated.Based on the analysis of Lpp protein sequences from different species of bacteria,we selected two strains of Enterobacteriaceae Kluyvera Georgiana and Pantoea agglomerans containing Lpp in their genomes,and Psychrobacter sp.,an altermonas bacterium that does not contain the Lpp gene in its genome.We extracted their cell walls and carried out a comparative study on the AFM.The results suggest that the granular structure observed in the gram-negative cell wall may be correlated with the presence or absence of the lpp gene in the genome.