| Aeromonas hydrophila is a Gram-negative bacterium widely distributed in various water environments and also is a typical primary conditional pathogen.The bacteria can secrete a variety of virulence proteins to infect host,causing lesions,diarrhea,vomiting,septicemia,cholecystitis and other clinical symptoms.Fish is particularly vulnerable to be infected by this pathogen.Current methods for the prevention and treatment of such fish diseases are largely accounted on antibiotics,but excessive use of antibiotics will inevitably increase the resistance of the bacteria,not only restricting the development of the aquaculture industry,but also increasing the prevention and control of bacterial infection.The difficulty of the disease will also cause serious damage to aquatic products and the ecological environment,thus threatening food safety and social public safety.Outer membrane proteins(OMPs)are proteins that are located on the outer membrane of gram-negative bacteria.They help the bacteria to intake necessary nutrients,promote the growth of bacteria,and play a important role on the physiological and pathological mechanisms of bacteria.The synthesis,transport,and correct folding of outer membrane proteins require a complex process in which precursors with N-terminal signals are first synthesized in the cytoplasm,mediated through the inner cell membrane via the SEC system,and mediated by the chaperone(Skp,SurA).Under the action of the periplasmic space,the ?-barrel structure is finally folded and integrated into the outer membrane with the aid of the BAM(?-barrel assembly machinery)complex.It was reported that BAM complexes are generally composed of five subunits such as BamA,B,C,D and E,and play an extremely important role in the synthesis and secretion of outer membrane proteins.At present,the research on this complex is limited to only a few bacteria,and the related functions in other bacteria have yet to be further studied.In this dissertation,Aeromonas hydrophila ATCC 7966 was used as a research strain to study the effect of this strain on the important bacterial physiological functions by constructing bamA,B,C deletion strains and the succinylation modification of BamA protein.Firstly,using homologous recombination technology,after in vitro PCR amplification of the upstream and downstream homologous arms of bamA,B,and C,positive selection of chloramphenicol-selective resistance and negative SacB in two homologous recombinations undergoing homologous recombination during the rescreening process,the target deletion strains ?bamA,B,C were obtained.Subsequently,SDS-PAGE analysis,PCR,16s DNA sequencing and other technical methods were performed to conform the correction of the depletion strains.In order to understand the effect of key gene-deleted strains of BAM system on the important physiological functions of A.hydrophila,the present study firstly measured the minimum inhibitory concentrations(MICs)of the deletion strains against Oxytetracycline(Oxy),Ciproxacin(Cip),and Tetracycline(Tetracycline,Tet)and Nalidixic acid(Na).The results showed that when compared with the wild-type strain,AbamA increased resistance to these four kinds of antibiotics to a certain extent with the most significant resistance in Oxy;AbamB had a higher MIC for CIP and the MIC of AbamC to Na was elevated;The growth curves of the mutants and the wild-type strains under the treatment of these four antibiotics were measured.The results showed that the ?bamA-defcient strains showed different degrees of growth under Oxy,Cip,Tet,and Na stress,and ?bamB was only changed under OXY treatment,whereas ?bamC only differed in Na treatment.Moreover,we found that the bamA-C-deleted strain significantly reduced bacterial extracellular protease secretion activity while hemolytic activity appeared to be relatively enhanced.To further confirm that the above-mentioned changes in the phenotype of the deleted strain which may be related to the influence of the transmembrane protein transport after the BAM system is deleted,western blotting was used to study the expression of four outer membranes or membrane proteins such as A0KQZ1,A0KHF6,A0KQ46 and A0KHF7 in bamA-C deletion strains in cytoplasm and membrane protein fractions of the bacteria.The results showed that when compared with wild-type strain,the depletion of bamA,B,and C resulted in the the decreasing expression of membrane protein,and AbamA and AbamC were the most significant changes,suggesting BamA-C protein transport and regulation in bacterial membrane proteins.In addition,our previous proteomic study found that the K558 lysine site of BamA was succinylated.In order to further study the effect of succinylation modification sites on the physiological function of A.hydrophila,a rescued strain in bamA deletion strain was constructed by gene recombination,and site-directed mutagenesis was used to mutate the corresponding lysine modification site from 558 K to 558R.The results showed that the mutation of the BamA K558R had a significant effect on the resistance to oxytetracycline.Growth curve analysis also found that the point mutations had a low growth rate and a longer plateau time.In summary,in this dissertation,bamA,B,C deletion strains and site-directed mutagenesis of BamA and related rescued strains in A.hydrophila were constructed by molecular biology methods.Our studies have found that BamA,B,and C proteins affect bacterial resistance,hemolytic activity,extracellular proteases,and other physiological functions.It has also been found that the succinylation of BamA protein K558 plays an important role in bacterial resistance.In this way,our results provide a preliminary theoretical basis for further understanding the biological function of BAM system in Aeromonas hydrophila. |
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