| Quorum sensing(QS)is involved in the regulation of virulence and spoilage of food related harmful bacteria.Aeromonas,as gram negative species,is an important conditional pathogen and spoilage bacteria in aquaculture.An isolate of A.salmonicida AE03 with high spoilage ability was isolated from spoiled large yellow croaker(Pseudosciaena crocea).Previous studies have shown that AE03 could produce color by biosensor,suggesting that the bacteria have acylated homoserine lactones(AHLs)activity.However,the regulatory relationship between AHLs activity and spoilage ability in A.salmonicida was still few.In this study production and major species of AHLs during the different condition were analyzed,and AHLs encoding gene asaI was amplified and deleted.Spoilage,biofilm and stress resistance among wild strain,asaI-mutant and supplementation of exogenous C4-HSL were compared.Genes expression of asal mutant on spoilage and stress resistance related enzymes in A.salmonicida was analyzed based on the qRT-PCR.The aim of this study was to assess the effects of AHLs mediated QS activity on the spoilage and stress resistance of A.salmonicida AE03.The main results are as follows:AHLs signal molecules in A.salmonicida AE03 were detected by Chromobacterium violaceum CV026 biosensor and HPLC-MS/MS.The strain AE03 could induce CV026 to produce the purple response both at 28? and 4?,exhibiting a density-dependence.With the increase of NaCl concentration,AHLs activity gradually increased and reached maximum at the concentration of 1.5%NaCl.Five types of AHLs were detected in AE03 culture by HPLC-MS/MS analysis,in which N-butanoyl-L-homoserine lactone(C4-HSL)as a major signal molecule.The AHLs in AE03 reached the highest activity at 28? for 30 h,similarly to the results of CV026.Thermal treatment rapidly degraded the AHLs signal molecules in the supernatant of culture.With the extension of heating time,the diameters of purple circle of CV026 decrease rapidly,indicating the loss of AHLs activity.The AHLs activity of AE03 was reduced by weak acid and alkaline treatment,and the optimum pH for maintaining AHLs activity was pH 7.8.The sequence of asal gene of AE03 isolate was amplified using primers asal-F and asaI-R,and the product was sequenced.A.salmonicida AE03 asaI-mutant strain was constructed and verified by PCR amplification and sequence.The asaI-mutant failed to trigger violacein pigment in the biosensor CV026.LC-MS/MS was further confirmed that total AHLs decreased significantly,and C4-HSL and C6-HSL lacked in asal-mutant.The growth,spoilage phenotypes,biofilm formation,swimming and stress resistance were compared between wild strain,asal-mutant with or without supplemented exogenous C4-HSL at 28?.Compared with wild type,the production of trimethylamine(TMA),biogenic amino and protease significantly increased in asaI-mutant during the exponential and stationary phase,while the growth rate didn't differ.Swimming motility in asaI-mutant was comparatively stronger than that of AE03,whereas,asaI-mutdant resulted in the decrease of maturing biofilm.Furthermore,supplementation of exogenous C4-HSL in asaI-mutant restored the production of spoilage metabolites,protease and biofilm formation.Compared to AE03,TMA production in asal-mutant increased 100.2%,22.3%and 24.9%at 12 h,18 h and 24 h respectively.When exogenous 20?M or 40 ?M C4-HSL was supplemented in asaI-mutant,compared to the asal-mutant,TMA amount decreased by 15.5%,6.2%,8.5%,and by 52.7%,28.5%,27.0%at 12 h,18 h and 24 h,respectively.Similarly,biogenic amino(BA)in asal-mutant increased by 21.5%,22.4%and 23.2%at 12 h,18 h and 24 h compared to wild strain.When the supplemented of 20 ?M or 40 ?M C4-HSL,the BA levels of asaI-mutant were decreased by 16.3%,7.2%,6.5%,and by 28.6%,14.5%,16.4%respectively.Similarly,activities of extracellular protease in asaI mutant was significantly higher than wild strain,and decreased in asaI mutant adding exogenous C4-HSL in a concentration dependent.Biofilm formation reached the maximum at 28? for 12 h,and maturing biofilm in the asaI-mutant decreased 32.1%than that of wild strain by crystal violet staining.Confocal microscopy observed the biofilm thickness of wild and mutant strains was 52.52 ?M and 36.66 ?M during the maturation respectively.Stress resistance results showed that asal mutant strain enhanced the resistance against high temperature,H2O2 and high NaCl.The survival of wild and asaI-mutant were 46.8%and 54.1%at 50? for 12 h,respectively.The survival of AE03 wild type and mutant were 70.3%and 87.1%after 2 mM H2O2 treatment for 5 min,respectively.Similarly,the survival of wild and mutant were 50.5%and 59.8%,respectively when 30%NaCl treated for 90 min.Therefore,it was indicated that the production of TMA,biogenic amines,extracellular protease,swimming motility and resistance in asaI-mutant significantly increased,while the biofilm formation decreased.The supplementation of exogenous C4-HSL partially restored the production of spoilage metablites and protease in asal-mutant.Spoilage potential of AE03 and asaI-mutant with or without C4-HSL was investigated in the sterile fish juice(P.crocea)stored at 4?.The results showed that there was no significant difference among AE03 wild type strain and asaI-mutant without or with C4-HSL on the growth in fish juice,and reached the early stationary phase at 3 d.The amount of TMA and TVB-N increased slowly in initial 2 d,and increased rapidly at 3 d and reached the highest value at 5 d.For the sterile fish juice,AE03,asaI-mutant and supplemented with C4-HSL(20?M and 40 ?M),extracellular protease was 5.92,8.16,6.64 and 5.88U,and TMA was 22.81,33.18,30.19 and 26.70 mg/100 mL,respectively.The TVB-N value of four groups was 22.7,27.30,24.85 and 22.62 mgN/mL,respectively.The above results showed that the deletion of asaI gene promoted the production of spoilage enzymes and metabolites of AE03,and accelerated the process of spoilage in fish juice,while exogenous addition of C4-HSL reduced the spoilage of asaI-mutant.To investigate whether asaI/asaR system regulates the expression of spoilage enzymes and motility genes,the transcription of tor A,cadA,fliR,katG and asaR were analyzed using RT-PCR in AE03 wild strain and asal-mutant without or with exogenous C4-HSL during the different growth phase.The transcription of genes gradually increased at 18 h,and then slowly decreased at 24 h.The transcript levels of five genes in asal-mutant significantly increased compared with that in the AE03.The highest transcript levels of torA,cadA,fliR,katG and asaR genes in asaI-mutant were 2.29,2.29,1.69 and 9.86 fold at 18 h respectively(p<0.05),while the highest transcript levels of asaR was 2.65 fold(p<0.01)at 12 h.The transcription of five genes gradually decreased in asaI-mutant during the stationary phase,and only torA,fliR and katG levels in mutant were significantly higher than that in AE03 at 24 h(p<0.01).Meanwhile,when asal-mutant was supplemented with C4-HSL,all transcription of four genes markedly decreased,and gradually restored to the levels of wild strain,especially 40 ?M C4-HSL.Additionally,transcript levels of asaR,fliR and cadA in asaI-mutant with 40?M C4-HSL were lower than that in AE03 after 24 h incubation.In summary,A.salmonicida AE03 exhibited high AHLs activity both at 4? and 28?.C4-HSL in AE03 negatively affect some related genes of spoilage and resistance,resulting in the increase of spoilage potential and resistance,while C4-HSL positively regulate biofilm maturation.It was indicated that spoilage potential,biofilm formation and stress resistance were under the control of AHLs mediated QS. |
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