Preliminary Study On The Regulation Mechanisms Of Peptidoglycan Assembly-associated Enzymes In Lactococcus Lactis Under Acid Stress

As an important protective layer,peptidoglycan(PG)cell wall plays pivotal roles in regulating acid stress.To explore the relationship between PG growth and the acidresistance mechanism of Lactococcus lactis F44,we performed a series of studies as follows:(1)The relations between the PG synthesis and acid stress1)Influence of acid stress and PG-synthases(PGSs)overexpression on PG growth:TEM analysis was used to identify the phenotypic changes(thickness)of PG cell wall of acid-stressed strains and PGSs-overexpressing strains.The results showed that the thickeness of PG cell wall decreased with acid stress.Moreover,PGSsoverexpressing strains(murG,murC,ponA)were characterized with a thicker PG cell wall.2)Post transcriptional regulation of PGSs by sRNA under acid stress:On the basis of the sRNA deep sequencing data of L.lactis F44 under acid stress,sRNA263 was successfully predicted to be involved in the post transcriptional regulation of PGSs.After being verified by RNA-RNA report system and site-directed mutagenesis,sRNA263 could positively affect the translational level of the target genes such as murA,murC and mraY by protecting these target mRNAs from RNase.Furthermore,TEM analysis showed that sRNA263-overexpressing strain displayed a thicker PG cell wall.(2)The relationship between the PG hydrolysis and acid stress1)The effect of regulating PG hydrolases(PGHs)activity via increasing D-Asp amidation level on PG growth:Overexpression of the asnH gene could maintain PG integrity by increasing amidation level through disturbing PGHs activity.This structural alteration conferred the acid-resistance phenotype to engineered strains.More importantly,this engineered strain was chacterized with a higher nisin yield than the wild-type F44.Considering the fact that PG cell wall was a defining structure in bacterial kingdom,cell wall remodeling provided a promising strategy for enhancing bacterial robustness of industrial microbes.2)The effect of regulating PG hydrolases(PGHs)activity via protease degradation on PG growth:Excessive PGHs activity were detrimental to bacterial growth,especially under acidic conditions,thus,the PGHs must be strictly controlled.Using bacterial two hybrid(B2H),we succeeded screening out the Ylj J,which might interact with YjgB(?-D-glutamyl-L-lysyl endopeptidase).The Bi Fc method was performed to further verify the interactions between YljJ and YjgB.However,YljJ did not contain any conserved regions responsible for the protease activity.Therefore,it is speculated that YljJ may be an intermediate to mediate the degradation of YjgB by other proteases.