Molecular Mechanism On Oxidative Stress Tolerance In Listeria Monocytogenes During Biofilm Formation

The foodborne pathogen Listeria monocytogenes has capability topersist on surfaces in food-processing environments, which makes it moreresistant to environmental stresses. Once L. monocytogenes persists on foodcontact surfaces and forms biofilm, it is difficult to remove by conventionalcleaning methods or inactivate by disinfection. Subsequently, food productsmay become contaminated during processing, which may introduce newchallenges to food safety in the food industry.The increased attention has been paid to biofilm formation by L.monocytogenes since the high mortality rate associated with listeriosis（20-30%）. Most researches focused on physiological responses of L.monocytogenes in biofilm, while the molecular mechanism of biofilmformation is still unclear. Recently, some mutants with the decreasedcapability of forming biofilm have been identified by screening a libraryconstructed via transposon Tn917mutagenesis of L. monocytogenes4b G（serotype4b） in our lab. In order to better understand the mechanismunderlying biofilm formation and stress-responses in L. monocytogenes, theinactivated genes of the mutants were identified first in the present study.GB5showing reduced biofilm formation and sensitivity to oxidative stress was further characterized. In addition, six serotype4b strains and six serotype1/2a strains were selected to observe biofilm formation and oxidative stresstolerance from physiological and genetic response. The main results are asfollows:Firstly, the inactivated genes caused by transposon Tn917insertion wereidentified by inverse PCR （IPCR）, and the revertants were constructed toverify their functions on biofilm formation. Four pair sets of specific primerswere designed according to the sequence of Tn917for inverse PCR, and thesuspected PCR products were sequenced. BLASTN analyses indicated thatthe inactivated gene in mutant GB1was identical to gene LMOf2365₂324（encoding a conserved hypothetical protein）, that in GB5was identical toLMOf2365₁758（gltB, encoding the large subunit of glutamate synthase）and that in mutant GB8was identical to LMOf2365₁497（encoding a MerRfamily transcriptional regulator）. Thus these genes were named as lm.G₂324,lm.G₁758（gltB） and lm.G₁497. The results of southern blot assay andbiofilm recovery of revertants showed that lm.G₂324, gltB and lm.G₁497were solely responsible for the positive regulation of biofilm formation intheir corresponding mutants.Secondly, stress tolerance of biofilm formation mutants GB1, GB5, GB8and the wild-type was compared. Only mutant GB5yielded reducedoxidative stress tolerance compared with the wild-type strain, demonstratingthe involvement of the inactivated gene in GB5（gltB） in both biofilmformation and oxidative stress tolerance. Other environmental stress factorssuch as osmotic pressure （1%-10%NaCl）, low and high pH （2.5-7.5）,different growth temperature （4C,10C,37C,42C）, and induction ofautolysis （induced by0.05%Triton X-100） did not result in significant differences between the wild-type and mutant strains.Thirdly, mutants with deletions in gltB and its upstream gene gltC wereconstructed, and their phenotypes were observed. The temperature-sensitiveplasmid pAUL-A was usded to construct the deletion mutants△gltB and△gltC. Both△gltB and△gltC showed reduced biofilm formation andincreased sensitivity to three oxidant agents （H2O2, CHP, PAA） relative to thewild-type, implying their role in the regulation of biofilm formation as wellas oxidative stress tolerance.Fourthly, functional analysis was carried out on△gltB and△gltC withqRT-PCR and initial adhesion assay. In the wild-type strain, gltB and gltCexpression was induced approximately8-fold and14-fold, respectively, withexposure to H2O2, providing further evidence that their gene products may beinvolved in the response to oxidative stress. The results of qRT-PCR revealedthat the gltB or gltC was most likely affected through mutual regulation underoxidative stress. Initial adhesion under liquid flow was tested in an adhesionassay, and the mutants△gltB and△gltC exhibited decreased adherence toglass surface, indicating that cell surface characteristics may be altered in themutants. It is hypothesized that a LysR-type transcriptional regulator encodedby gltC plays a critical role in controlling expression of genes involved inoxidative stress tolerance, surface attachment and biofilm formation.Fifthly, the genes except for gltB and gltC related with biofilm formationas well as oxidative stress tolerance in L. monocytogenes were analyzed, andthe molecular mechanism was explored. Twelve strains from two commonserotypes （1/2a and4b） of L. monocytogenes were selected for furthercharacterization on biofilm formation and oxidative stress tolerance. Most1/2a strains exhibited a relatively hyper-biofilm than the4b strains in test conditions, while the later demonstrated a stronger tolerance than the former.Interestingly, in L. monocytogenes4b strains, it seems that there was apositive correlation between biofilm-formation and oxidative-stress-response,while no connection in1/2a strains. In a qRT-PCR assay, threeoxidative-stress-related genes （sod, fri and perR） displayed their importanceof serotype variation in stress resistance and the recA gene appeared to becritical for survival of L. monocytogenes in biofilm.Overall, three novel genes were identified to be involved in positiveregulation of the biofilm formation by L. monocytogenes in this study, whichare named lm.G₂324, gltB and lm.G₁497. Among them, the gltB gene（encoding the large glutamate synthase）, mutually regulating its upstreamgene gltC,（encoding a LysR-family transcriptional regulator of the gltBDoperon）, was responsible for oxidation resistance and biofilm formation in L.monocytogenes4b G. The gltC gene might play a critical role in controllingexpression of genes involved in oxidative stress tolerance, surface attachmentand biofilm formation. Except for gltB and gltC, other four genes sod, fri,perR, recA showed the importance for the oxidative stress tolerance andbiofilm formation in L. monocytogenes serotypes4b and1/2a strains. Inaddition, there was a positive correlation between biofilm formation andoxidative stress response in L. monocytogenes4b strains.