Preliminary Machanism Research Of A New AlgC-type Protein Le2152 Regulation On Twiching Motility In Lysobacter Enzymogenes

OH11 is a promising agricultural biocontrol bacteria isolated from capsicum rhizosphere soil which has a wide range of antifungal activities.It not only produces a variety of extracellular hydrolases,but also secrets the antifungal substance HSAF(Heat-Stable Antifungal Factor)to control plant diseases caused by various plant pathogenic oomycetes and fungi.Twiching motity mediated by T4P(type Ⅳ pili,T4P)is an important classification basis of Lysobacter enzymogenes,and also determines the adsobption,colonization and other activities of Lysobacter enzymogenes also need twiching motity,thus it is also regarded as an important factor to evaluate the biocontrol ability of Lysobacter enzymogenes.In this paper,we use Lysobacter enzymogenes strain OH11 as a working model to explore the regulatory mechanism of its twiching motity.We previously studied T4P(type Ⅳ pili,T4P)system and reported PilR and Clp as key regulators in twiching motity.In order to explore new regulatory mechanism,we screened the mutant library of OH11,and got a strain without twiching motity,which was identified as Le2152 gene defective mutant.We analyzed the domain of Le2152 in Pfam and found Le2152 contains PGM-PMM(phosphoglucomutase-phosphomannomutase)domain.In the OH11 genome,there was another protein Le4861 also contains this domain,but there was no influence on the twiching motity after Le4861 knockout.In order to explore the reasons for the diffierence between Le2152 and Le4861.we use the bioinformatics software TMHMM and SMART to analyzed and predicted their domain.The results showed that in addition to PGM-PMM domain,Le2152 has a cytoplasmic dCache domain on the N-terminal,and this domain is different from several previously reported dCache domains.The proteins both contain domin like Le2152,existing at least 8 orders of gammaproteobacteria,which means Le2152 are conservative and representative.After the first cloning of enzy matically active soluble of Pseudomonas aeruginosa AlgC in 1991,In the past,AlgC-type protein was studied by N-terminal truncation ORF.This study proved that Le2152 protein in Lysobacter enzymogenes not only has PGM-PMM domain,but also has dCache domain to form a complete ORF,and we detected the same N-terminal cytoplasmic domain as Le2152 in Pseudomonas aeruginosa PMMs.Le2152-type phosphomannosemutase can represent a new AlgC-type protein.In order to find out the mechanism of Le2152 in regulating twiching motity,we first screened the interaction proteins of Le2152 by Co-IP technology,and identified 403 potential interacting proteins.A preliminary analysis revealed that there were 7 proteins belongs to the TP4 system controlled twiching motity.Subsequently,we use bacteria two hybrid system to test the 7 proteins we picked up in Co-IP data and finally found that PilA and PilS interact with the Le2152 protein.Next,we truncated Le2152,PilA and PilS,then we found that the interaction between Le2152 and PilA/PilS occurred in the transmembrane domin.In order to prove the specificity of this interaction,we analyzed all transmembrane proteins in Co-IP data,42 transmembrane proteins were found,and then we selected 5 of them to test with Le2152 by bacteria two hybrid system.We found that only Le3673 can interact with Le2152,which means not all transmembrane proteins interact with Le2152.Then we selected the PilA protein to explore the possible regulatory pathway of Le2152.We found that Le2152 does not regulate PilA by affecting the expression,secretion and localization of PilA protein,so the specific regulatory pathway of its interaction needs to be further explored.