Effects Of Different Lengths Of Braun’s Lipoprotein On The Physiology Of Escherichia Coli

A significant difference in cell structure between Gram-negative bacteria and Grampositive bacteria is that their cell wall has an outer membrane structure in addition to peptidoglycan.A stable outer membrane structure provides a stable periplasmic space for Gramnegative bacteria,thereby ensuring the normal progress of many important life activities in the periplasmic space.In E.coli,Braun’s lipoprotein Lpp is one of the most important proteins performing the function of stabilizing the outer membrane structure.The Lpp protein has a small molecular weight and a single-strand α helix in structure,often existing in the form of a trimer.Lpp is also the only outer membrane protein that can be covalently linked to the peptidoglycan layer in E.coli.Its N-terminus is linked to three fatty acid molecules and inserted into the outer membrane,and its C-terminus is covalently linked to the peptidoglycan layer of the cell wall.superior.An E.coli cell contains hundreds of thousands of Lpp molecules,which jointly support the outer membrane and ensure the stability of the outer membrane structure.In addition,Lpp also participates in a series of important life activities in the periplasmic space.As confirmed by the previous research of our research group,Lpp also plays an important role in the process of E.coli cell division.To adapt to different living environments,bacteria in nature have evolved Lpp of different lengths.The length of Lpp directly affects the distance between the outer membrane and the peptidoglycan layer,which in turn affects the height of the periplasmic space.Studies have shown that after lengthening and truncating the length of Lpp protein,the height of the periplasmic space of E.coli will change accordingly,and some life processes in the periplasmic space will also change accordingly.For example,the change of Lpp length will affect the mechanical properties of the bacteria,change the assembly of important protein complexes in the periplasmic space,affect the information transmission across the periplasmic space,and so on.In this paper,based on the existing work in the laboratory,mutant strains of E.coli with different lengths of Lpp were constructed,and the effects of changing the length of Lpp on the physiological functions of E.coli were studied.The specific research contents are as follows:1.Construction of Lpp truncated E.coli mutant strainsThe E.coli lpp-12 and lpp+21 mutant strains with 21 amino acid residues truncated and 21 amino acid residues increased were constructed.Using AlphaFold2 to predict the structure of the transformed Lpp,it is preliminarily judged that the constructed protein can be folded into a normal single-strand α-helical conformation.The mutant gene constructed by RT-qPCR can be normally transcribed.The peptidoglycan layer of the mutant cell wall was extracted,and atomic force microscopy was used to confirm that the mutated Lpp protein could be correctly positioned and covalently bound to the outer surface of the peptidoglycan layer.Under the atomic force microscope,the Lpp particles on the peptidoglycan layer of the lpp-21 mutant became smaller.which may be caused by the difficulty in forming trimers after Lpp truncation.Moreover,the peptidoglycan layer of the lpp-21 mutant strain had a large number of holes formed by the lack of Lpp.2.The effect of modified Lpp length on cell morphology and growth stateTaking E.coli wild-type WT strain and Lpp-deleted Δlpp mutant strain as controls,it was found that the growth curves of E.coli lpp-21 and lpp+21 mutant strains with modified Lpp length were similar to those of WT strain when cultured under standard culture conditions,indicating that the growth of the mutant strains was not significantly affected.Using optical microscope,transmission electron microscopy,atomic force microscopy,etc.to study the morphology of the mutant strains,it was confirmed that the mutant strains can grow and divide normally,and the morphology of the mutant strains has not changed greatly.However,the lpp-21 mutant strain produces a large number of pili.It is speculated that the expression of a large number of pili may cause a hole structure formed by the lack of Lpp on the peptidoglycan layer.3.Effects of modified Lpp length on the physiology and environmental adaptation of mutant strainsA fter the length of Lpp protein was changed,the tolerance of lpp-21 and lpp+21 mutants to NaCl concentration decreased to some extent.The sensitivity of the lpp-21 mutant to EDTA was similar to that of the WT strain,but the lpp+21 mutant was very sensitive to EDTA.After the length of Lpp was changed,the tolerance of the two mutants to the high-pressure growth environment decreased.The swimming and swarming abilities of lpp-21 and lpp+21 mutants w ere weaker than those of WT strain;the biofilm formation ability of lpp-21 mutant was similar to that of WT strain,but the biofilm formation ability of lpp+21 mutant was enhanced.The resistance of the two mutants to antibiotics was similar to that of the WT strain,and only a few antibiotics such as Fosfomycin had weakened antibacterial effects on the lpp-21 mutant.The above studies have revealed the effects of altered Lpp protein length on the morphology and physiological functions of E.coli.The work in this thesis contributes to a deeper uunderstanding of the significance of Lpp in maintaining the physiological functions of E.coli and provides an important basis for further studies on the physiological functions of Lpp.