Molecular Mechanisms Of Acetobacter Pasteurianus In Acetic Acid Resistance

Acetic acid bacteria?AAB?is widely used in vinegar manufacture due to its robust ability in oxidizing ethanol to produce acetic acid.As a critical factor in the production of vinegar,the mechanisms of acetic acid resistance in AAB have been elucidated gradually during past decades via proteomic and genomic analysis.However,the involved molecular regulating networks were still poorly understood to date.In the current study,Acetobacter pasteurianus Ab3 isolated from Zhejiang traditional rose vinegar was adopted to unravel the resistant mechanisms to acetic acid,emphasizing on the regulating and responsive networks.Quorum sensing as a kind of regulating model is widespread in bacteria.To investigate the existence of QS in A.pasteurianus,two strains Ab3 and CICC 20001 were used firstly.The results showed that the well-known signal molecules such as N-acyl-homoserine lactones?AHLs?and Autoinducer-2?AI-2?were not detected in these two strains,which was consistent with the loss of genes coding for AHLs and AI-2 synthases and receptors by genome analysis.Moreover,this gene loss phenomenon universally existed within the A.pasteurianus strains tested.Contrast to that of A.pasteurianus,the LuxI and LuxR homologs were widely found within Komagataeibacter and Gluconacetobacter strains,implying that the QS function actively.In addition,the QS systems which compile the other signal molecules such as diffusible signal factors?DSF?,and quinolone signal?PQS?were not found in A.pasteurianus genomes.It implies that A.pasteurianus can't utilize the known QS to modulate its physiological process.The isobaric tags for relative and absolute quantification?iTRAQ?analysis was performed to investigate the potentially regulatory modules further.Consequently,1386 proteins were identified totally,and more than 150 kinds of proteins were shown to be differentially expressed under circumstance with high acid concentration?p<0.05?.Critically,four main biological processes,including ribosome and protein metabolism,amino acid metabolism,fatty acid biosynthesis,and oxidative stress responding,were concluded to participate in cell homeostasis maintenance for resisting on acetic acid stress.Interestingly,two-component systems and toxinantitoxin systems were detected firstly to be relevant with acid resistance in A.pasteurianus,which tells us a new clue that TAS may serve as the main modulatory network within A.pasteurianus.By bioinformatics analysis of A.pasteurianus Ab3 genome,21 putative type II TASs were predicted.Three putative TAS candidates?DB34₀3205–DB34₀3210,DB34₀4120–DB34₀4125,and DB34₀1190–DB34₀1195?were selected and characterized further.The genetic structure analysis showed that DB34₀3205–DB34₀3210 and DB34₀4120–DB34₀4125 had the typical structure of type II TAS,and both of the toxin and antitoxin gene shared a same promoter.Protein domain analysis showed that DB34₀3205–DB34₀3210 and DB34₀4120–DB34₀4125 had the conserved domain of HicAB and HigAB,respectively.While DB34₀1190–DB34₀1195 didn't possess the canonical structure of type II TAS.Further the heterogeneous protein expression analysis showed that the three putative TASs had activities similar to the classical type II TAS.Thus,DB34₀3205–DB34₀3210 and DB34₀4120–DB34₀4125 were named as Ap_hicAB and Ap_higAB,respectively,while the atypical DB34₀1190–DB34₀1195 was named as Ap_npoTA.Beyond,the prevalence and diversity of type II TAS in AAB were linked to the bacteria biotopes tightly.Specifically,A.pasteurianus strains,universally domesticated and utilized for industrial vinegar manufacture,contained a higher amount of type II TA loci?>10?than that of the host-associated ones?<5?,suggesting that TAS may play a underlying role in cell adaptation to acetic acid stress.Furthermore,toxins RelE and PIN_VapC,and antitoxins RHH and AbrB domains were the principal hubs of the TAS networks in A.pasteurianus.Population heterogeneity are developed universally in the bacteria growths.In terms of physiological variants,persister is another unique and characteristic subpopulation in parallel with VBNC.Here we firstly explored the existence of persister of A.pasteurianus Ab3 in vinegar fermentation.During the different growth phases,A.pasteurianus Ab3 performed persistence formation.Specifically,the persister ratio in exponential growth phase reached up to 1%,which was much higher than that of the other bacteria such as Escherichia coli?<0.1%?,suggesting the persister formation help to confer the acid stress to A.pasteurianus.By increasing the ATP synthesis,ethanol and acetic acid negatively influenced the persister formation.Beyond A.pasteurianus,the persister formation were observed in other AAB strains also.In order to unravel the link between the TAS and acetic acid resistance of A.pasteurians,the function of Ap_hicAB was investigated.Meanwhile,a set of gene manipulation tools specifically suitable for A.pasteurianus were constructed.The results showed that the disruption of Ap_hicAB significantly reduced the producing rate and final accumulation of acetic acid by A.pasteurianus Ab3.Also,the persister formation decreased remarkably.Furthermore,the transcriptional level of Ap_hicAB was found to be stable in condition with low acidic titer,but increase as much as 10 to 20 fold under high acid stress,which suggests that Ap_HicAB is functional in the stress adaptation of A.pasteurianus.To further elucidate the possible pathway regulated by Ap_HicAB,40 genes involved in different biological processes were selected for qRT-PCR analysis.Compared to that observed in Ap_hicAB deficient mutant,more than 80% genes tested above in wild type strain were up-regulated at the transcription level,indicating a positive regulatory role of Ap_HicAB in A.pasteurianus.Twelve genes with positive two-fold involved in amino acid metabolism,protein biosynthesis and metabolism,and two-component systems etc.Notably,the TAS Ap_higAB and Ap_npoTA were up-regulated?>2.5-fold?,implying the cooperation among different TASs.Collectively,Ap_HicAB is suggested to play an important role in regulating different bioprocesses upon unfavorable conditions,thereby benefit the cell growth.In conclusion,the mechanisms of acetic acid resistance in AAB are intricate.With the development of systems biology and evolutionary biology,the application of multiple omics will expand our knowledge about the acid resistance mechanisms,especially the regulating networks,in A.pasteurianus.This will help the construction and breeding of appropriate AAB strains,and further enable the improved fermentation conditions and upgraded vinegar industry.