The Subcellular Location Of Protein Encoded By Newly Found MucA Mutation Gene, The Influence Of The Protein On Biofilm Formation And The Mechanism Of The Influence

Objective:To investigate the subcellular localization of the protein encoded by newly found mucA mutation gene (mucA56), observe the influnence of the mucA56 gene on biofilm formation and structure, and further explore the mechanism of the influnence.Methods:mucA56 gene is amlified from pMD18T-HR with Ncol and Xbal restrictive endonuclease site and cloned into pMF54 expression vector, resulting in pCAQ56; alkaline phosphatase gene fragment resulting from Xbal digesting of pPHO7 is fused to the open reading frame (ORF) of pCAQ56, resulting in the mucA56-phoA fusion expression vector pCAQ57; Gene fusion vector pCAQ57 is confirmed first by PCR and sequencing, then by western blot using anti-alkaline phosphatase antibody. pCAQ57, pKMG170 (negative control plasmid), pKMG176(positive control plasmid) are transformed into Ecoli. DH5a and Pseudomonas aeruginosa PAO1, then transformants are cultured on the LB agars containing alkaline phosphatase substrate BCIP, which could evaluate the activity of alkaline phosphatase; A enhanced green fluorescent protein gene is amplified from pGFPuv and cloned into E.coli-P.aeruginosa Shuttle vector pUCP20, resulting in a green fluorescent protein expression vector pUCP20/GFPuv which can label P.aeruginosa; pUCP20/GFPuv are transformed into P. aeruginosa PAO1, PDO300 (the mucoid PAO1 derivative with classic mucA22 mutation) and PAOmucA56 (the mucoid PAO1 derivative with new mucA56 mutation) by electrotransformation. Biofilms with 1-day-aged, three-day-aged and five-day-aged formed by GFP labeled P. aeruginosa are observed by laser scanning confocal microscope. The P.aeruginosa biofilm cells with 1-day-aged, three-day-aged and five-day-aged were collected, total bacterial protein extract of which are then gone through two-dimensional electrophoresis. The protein spots which represent the same expression level between PAO and PAOmucA56 but different from PDO300 are picked up for further identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF MS) analysis. Results:The constructed mucA56-phoA fusion expression vector pCAQ57 is confirmed right by PCR and sequencing,WB further confirmed the expression of hybrid proteins. The colony colour of E. coilDH5a(pCAQ57), PAO1(pCAQ57), E. coliDH5a(pKMG170) and PAO1(pKMG176) on LB plate containing BCIP are all white, indicating no activity for the alkaline phosphatase. Observation of the biofilms with 1-day-old, three-day-old and five-day-old of PAO1,PAOmucA56 and PDO300 by Laser scanning confocal microscope reveal that PAO1 and PAOmucA56 biofilm develope from a uniform monolayer of attached cells into a biofilm characterized by an almost complete substratum coverage and even biomass distribution, the average thickness of the PAO1 and PAOmucA56 biofilm is 30μm and 25μm respectively; PDO300 forms a significantly different biofilm architecture, with attached cells growing exclusively in discrete microcolonies resulting in a low substratum coverage and high structural heterogeneity, the maximum thickness of PDO300 is 36μm; Proteomes of total protein extract of the biofilm bacterial with 1-day-aged, three-day-aged and five-day-aged reveals that the number of the protein spots whose abundance are same between PAO1 and PAOmucA56 but different from PDO300, were 8,6 and 15, respectively. Mass spectrometry results show the reliability of twelve proteins are not high. Among the seventeen candidate proteins, DNA directed RNA polymeraseβsubunit, glutamine synthetase, endopeptidase Clp2, elongation factor Tu, DNA directed RNA polymerase a subunit, elongation factor G1, heat shock protein HtpG, ATP dependent Clp protein ATP binding subunit clpX, trigger factor are relatively down-regulated in PDO300; 30S ribosomal protein S18, Phosphoenolpyruvate carboxykinase, Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, PasP, Biotin carboxylase, Arginine deiminase,Ornithine carbamoyltransferase, Serine hydroxymethyltransferase 3 are relatively up-regulated in PDO300; Especially,one clp protease family (Endopeptidase Clp2 and ATP dependent Clp protein ATP binding subunit clpX)is relatively down-regulated in the third day and fifth day of biofilm bacteria in PDO300, respectively. Conclusion:The MucA56 protein encoded by mucA56 gene is localized in the cytoplasm; Alginate might affect the biofilm formation, however, mucA56 gene influence the biofilm formation in another pathway other than alginate; mucA56 gene is a pleiotropic regulatory gene,which regulate many genes including genes involved in bacterial stress response, alginate synthesis and so on.The differentially expression of clp protease family may be the reason of the special biofilm morphology of PAOmucA56.