Functical Identification Of Importance Membrane Proteins In Klebsiella Pneumoniae

Membrane proteins are critical for cellular activities and account for approximately one-third of the entire proteins in cytomembrane.Membrane proteins participate in myriad biochemical events including substance and energy transportation,signal initiation and transduction,infection and immunity,response to external stimuli,catalysis and maintenance of membrane structure.In addition,some of membrane proteins are well-tailored virulence factors.To date,membrane proteins have received much more attention because they are potential in the field of drug targets,signal receptor and nano-channel.In fact,more than 70%membrane proteins are drug targets,indicating their correlation and importance in pharmaceutical engineering and biotechnology.This present study focused on the structural and functional identification of several membrane vital proteins in Klebsiella pneumoniae.Firstly,the K.pneumoniae membrane proteins were subjected to bioinformatics analysis.Based on spatial locations in cytomembrane,membrane proteins are roughly divided into two groups:inner membrane proteins and outer membrane proteins.The inner membrane proteins govern the transportation of substances and energy as well as information exchange between cells and their surroundings.The outer membrane proteins serve as receptors,enzymes,surface antigen,etc.To portray a picture of the membrane proteins in Klebsiella genera,the research advances were overviewed,and the functions of K.pneumoniae membrane proteins were analyzed through data mining of NCBI and KEGG,as well as gleaning of literature.Specially,the transmembrane region of K.pneumoniae membrane proteins was predicted by TMHMM server v.2.0 software,and the highly conserved genes were subjected to sequence alignments.Considering membrane proteins are macromolecule and contribute largely to membrane skeleton,their functions were identified by knocking out the coding genes.Deletion of genes led to three mutants named K.pneumoniae,K.pneurymoniae ?pspA,K.pneumoniae ?pspD and K.pneumoniae ?psp A ?pspD.The genes pspA?KPN₀1297?and pspD?KPN₀1300?are key for K.pneumoniae,because they could affect the storage and feedback the engery call PMF which produced by electrochemical gradient respiratory quinone.The power potential of proton is the driving force of ATP synthesis.SDS-PAGE analysis was performed to investigate the effects of pspA and pspD genes on protein expression levels of K.pneumoniae.Three differential bands of 100-130 KDa,100 KDa and 35-40 KDa were observed on the gel of K.pneumoniae.The three bands were further analyzed by MALDI-TOF/TOF-MS and fluorescence quantitative PCR.Results showed that the three enzymes are associated with glucose metabolism,outer membrane proteins and porins.Furthermore,western blot analysis showed that the ompF gene in K.pneumoniaeApspD?pspA was down-regulated compared with that in wild type.To determine the effects of gene deletion on surface morphology,SEM and AMF analysis was performed.Results showed that the mutants manifested less wrinkles than wild type.However,the difference is slight.In addition,shake flask cultivation of these strains was to investigate the biomass and carbon source consumption using glycerol or glucose as carbon source.Results showed that both the glycerol consumption and biomass of K.pneurmoniaeApspA are higher that of wild type K.pneumoniae,indicating that the genes pspA and pspD affect metabolism.