Investigation Of Acid-tolerant Mechanisms And Genetic Stability Of Acetobacter Pasteurianus CICC 20001 And CGMCC 1.41 Based On Genomics

Acetic acid bacteria(AAB)are a group of gram-negative obligate aerobic bacteria,which can oxidize ethanol,sugar or sugar alcohol to corresponding organic acids such as acetic acid,gluconic acid and indole-3-acetic acid in presence of oxygen.Up to the beginning of 2016,17 genera,including Acetobacter,Acidomonas,Ameyamaea,Asaia,Bombella,Gluconobacter,Gluconacetobacter,Granulibacter,Komagataeibacter,Kozakia,Neoasaia,Neokomagataea,Nguyenibacter,Saccharibacter,Swaminathania,Swingsia and Tanticharoenia,total about 85 species have been recorded in AAB group.AAB have been widely used to produce acetic acid,cellulose,indoleacetic acid,surfactant and pigments,and some AAB strains are even pathogenic.However,producing acetic acid may be the main application of AAB.Among AAB,Acetobacter and Komagataeibacter are main AAB genera that have been widely used to brew vinegar.The former are mainly applied to brew cereal vinegar in the East,while the later contribute to brewing fruit vinegar in Europe and the United States.Acetobacter pasteurianus(Ap)CICC 20001(once named Huniang 1.01)and CGMCC 1.41(once named AS 1.41)may be the earliest screened AAB isolates in China,and are still widely used to brew vinegar,displaying high genetic stability and high abilities in acetic acid production and tolerance.In vinegar industry,how to increase the ability of acetic acid production and tolerance,and how to maintain their genetic stability is of great importance.Currently,it is believed that acetate assimilation,transportation systems,cell membrane compositions,stress proteins expression,and so on,are related to acetic acid resistance in AAB.However,researches on mechanisms conferring acid resistance in Escherichia coli and lactic acid bacteria show that some mechanisms,such as chloride transporters,arginine-dependent systems,glutamate-dependent systems,also contribute to acid resistance in microorganisms.In addition,in terms of genetic stability in acetic acid production and tolerance in AAB,K.europaeus have been recognized but Ap exists a large controversy.This study investigated effects of conditions on acetic acid fermentation by Ap CICC 20001 and CGMCC 1.41,and then they were subjected to SMRT sequencing by using a PacBio RS DNA sequencer.Based on comparative genomics,mechanisms conferring acetic acid tolerance and genetic stability in Ap were investigated.Finally,the probable effects of DNA methylation on regulating acetic acid production and tolerance in AAB were studied.1 Effects of conditions on acetic acid production by Ap CICC 20001 and CGMCC 1.41.Numerous studies showed that adding some initial acetic acids promoted acetic acid production significantly.Therefore,based on liquid-state fermentation in a shaker(GYP medium,250 mL Erlenmeyer flask containing 100 mL medium,170 r/min,30?,6%ethanol),effects of different types and concentrations of initial acid on acetic acid production by Ap CICC 20001 and CGMCC 1.41 were investigated.The results showed that adding 1%and 0.5%initial acetic acid promoted acetic acid production by Ap CICC 20001 and CGMCC 1.41,respectively.While adding more than 1.5%and 3.5%initial inhibited their fermentation;The promotions of adding 1%and 0.5%other organic acids,including succinic acid,citric acid,oxalic acid,malic acid and lactic acid,were far from those of isocyatic acetic acid.Furthermore,the isocyatic oxalic acid,malic acid and lactic acid inhibited acetic acid production significantly.When the pH values of media were adjusted to 3.86 and 3.54(equivalent to 0.5%and 1%acetic acid pH values)with HCl,acetic acid fermentation by CICC 20001 and CGMCC 1.41 were remarkably inhibited.It suggested that the promotions of adding initial acetic acid derived from acetic acid itself,but the effects on modifying pH values.Further analysis showed that when no acetic acid was added,both of alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH)activities were approximately 0.3 U/mL.Whereas,when 1%and 0.5%initial acetic acid was added,ADH and ALDH activities were increased by 15%to 20%.It indicated that adding initial acetic acid promoted acetic acid production through increasing ADH and ALDH activities.Effects of different ethanol concentrations on acetic acid production by CICC 20001 and CGMCC 1.41 were investigated in the media containing 1%and 0.5%acetic acid,respectively.The results showed that adding 6%and 8%initial ethanol were best for acetic acid production by CICC 20001 and CGMCC 1.41.And the final acetic acid reached 6.25 g/100mL and 6.75 g/100 mL,respectively.However,when the initial ethanol was more than 6%and 8%,adaptive phase increased a lot,the final acetic acid not increased anymore,and percent conversion of ethanol decreased.In the media containing 1%and 0.5%acetic acid,as well as 6%and 8%ethanol,adding 0.5%arginine promoted acetic acid production by Ap CICC 20001 and CGMCC 1.41,and the rate of increase reached 2.1%-3.3%;Adding 1 mmol/L Mg2+and K+could promote acetic acid production,too,while the isocyatic Fe3+and Zn2+inhibited acetic acid production;When 50 mL media were added into 250 Erlenmeyer flask,Ap CICC 20001 and CGMCC 1.41 produced acetic acid up to 6.8 g/100mL and 7.35 g/mL,and the ADH activities reached 0.42 U/mL and 0.38 U/mL,respectively.In a word,promotions of initial acetic acid on acetic acid production by Ap CICC 20001 and CGMCC 1.41 derived from acetic acid itself,but the effects on modifying pH values;The maximum initial ethanol concentration was 6%and 8%for Ap CICC 20001 and CGMCC 1.41 fermentation,respectively;1 mmol/L Mg2+and K+promoted acetic acid production,but the isocyatic Fe3+and Zn2+inhibited acetic acid production;0.5%arginine also boosted acetic acid production;50 mL media in 250 Erlenmeyer flask was beneficial to acetic acid production.2 Genome characteristics and acid-tolerant mechanisms of Ap CICC 20001 and CGMCC 1.41Genomes of Ap CICC 20001 and CGMCC 1.41 were sequenced,assembled and annotated by using the third-generation sequencing technology.Their genomes both were assembled into 1 complete chromosome and several plasmids(10 plasmids in Ap CICC 20001 and 7 plasmids in CGMCC 1.41).Their genome size was 2865612 bp and 2928931 bp,G+C%was 52.94%and 52.95%,and the predicted coding sequence was 3006 and 3012,respectively.Comparison of genomes from 9 sequenced Ap strains showed that Ap chromosomes shared a high homology,but their plasmids varied.It suggested a separate evolution between Ap chromosomes and plasmids.The results also showed that amino acid compositions in all Ap strains were similar,containing a large amount of alanine,leucine and glycine.As to mechanisms conferring acetic acid resistance,based on comparative genomics,the results showed that the copy numbers of PQQ-ADH was in proportion to the ability to produce acetic acid in AAB;Ap CICC 20001 and CGMCC 1.41 genomes contained all of the genes related to the discovered mechanisms conferring acetic acid resistance in AAB,including acetate assimilation,transportation systems,cell membrane compositions and stress proteins expression.In addition,like in Escherichia coliy membrane cyclopropane fatty acids might be related to acetic acid production and tolerance in Ap;By the effects of decarboxylase,threonine,asparagine and ornithine were transformed into ammonia,and might contribute to acetic acid production and tolerance in Ap,too.In brief,based on genome sequencing and comparative genomics,the results showed that basic genome features,such as genome size and composition,were similar;Chromosomes were relatively conservative,but plasmids varied remarkably.Ap CICC 20001 and CGMCC 1.41 genomes contained all of the genes related to the discovered mechanisms conferring acetic acid resistance in AAB.In addition,membrane cyclopropane fatty acids and the catabolisms of threonine,asparagine and ornithine might contribute to acetic acid production and tolerance in Ap,too.3 Investigation of AAB genetic stabile differences based on comparative genomics.Currently,genetic stability in acetic acid production and tolerance in Ap exists a large controversy.Therefore,distribution of factors related to genetic stable and instable were investigated in the relatively instable Ap strains,including 8 Ap IFO 3283 substrains,and the relatively stable Ap strains,including CICC 20001,CGMCC 1.41 and 386B.In bacteria,insert sequence,genomic islands,prophage,plasmids,and so on,were main factors related to genetic instability.While,restriction-modification system,toxin-antitoxin system and CRISPR-Cas system were main factors contributing to genetic stability.The results showed that Ap IFO 3283 substrains possessed 156 instable factors,but 386B,CICC 20001 and CGMCC 1.41 had 101,117 and 148 instable factors,respectively.Meanwhile,Ap IFO 3283 substrains owned 5-6 stable factors,but 386B,CICC 20001 and CGMCC 1.41 had 4,5 and 8 stable factors.The above analysis showed that comparing to instable IFO 3283 substrains,stable 386B possessed less stable factors and instable factors,while CICC 20001 and CGMCC 1.41 had less instable factors and more stable factors.Therefore,in Ap strains,the difference of genetic stability in acetic acid production and tolerance might be related to the quantity and composition of stability factors and instability factors.4 Probable effects of DNA methylation on acetic acid fermentation by Ap CICC 20001 and CGMCC 1.41 based on their genomesGenomes of 200 strains from virus,archaea,bacteria and eukaryote were selected to construct phylogenetic trees of m6A,m4C and m5C methyltransferase.The results showed that m6A methyltransferase were widely distributed in archaea and bacteria,and rarely existed in eukaryote;M4C methyltransferase were widely distributed in archaea,bacteria and eukaryote;M5C methyltransferase were widely distributed in eukaryote,and rarely existed in bacteria;No DNA methyltransferase were found in viruses.Further analysis showed that 61 m6A methyltransferases,62 m4C methyltransferases and 5 m5C methyltransferases were distributed in 24 AAB whose genomes had been sequenced and published.Each AAB had at least 1 m6A and m4C methyltransferase.It indicated that m6A and m4C were main DNA methyltransferases in AAB.Ap CICC 20001 had 6 m6A methyltransferases,6 m4C methyltransferases and 1 m5C methyltransferase,while CGMCC 1.41 possessed 6 m6A methyltransferases,3 m4C methyltransferases and 1 m5C methyltransferase.The analysis also showed that m6A in Ap belonged to y type-"motif ?-?-?-?-?-?-?-TRD" which the putative target recognition region(TRD)was located at the terminal of motifs,while m4C in Ap belonged to ? type-"motif ?-?-?-?-?-TRD-?-?-?-?" which TRD was situated at the middle of motifs.Among AAB,only Ap CICC 20001,Ap CGMCC 1.41,Gluconobacter oxydans 621H,Gluconacetobacter diazotrophicus PAI5 and Komagataeibacter xylinus E25 possessed a DNMT1 belonging to m5C methyltransferase and was related to gene silencing of most genes.Based on the methylation information from the third-generation sequencing,distribution of DNA methylation motifs in Ap CICC 20001 and CGMCC1.41 chromosomes were investigated.The results showed that both Ap CICC 20001 and CGMCC 1.41 contained motif "GTAC" and "GANTC"."GANTC" was target motif of most CcrM which was a well-known m6A methyltransferase contributing to regulate cell-cycle,while the function of "GTAC" was unclear.Ap CICC 20001 also had two unpublished motifs,"AACGAG" and "TGGCCA".The motifs were mapped to the genes related to acetic acid production and tolerance in Ap.The results showed that in CICC 20001,more than 95%genes that were related to acetic acid production and tolerance possessed motifs"GTAC" and "GANTC" and the motifs were usually located at the upstream of genes;In CGMCC 1.41,more than 96%had motif "GTAC",and a small number of genes contained motif "GANTC".In addition,other motifs were also distributed in genes related to acetic acid production and tolerance.It was probable that DNA methylation,as well as DNA methyltransferase,contributed to acetic acid production and tolerance.