Genetic characteristics of cellulose-forming acetic acid bacteria identified phenotypically as Gluconacetobacter xylinus

Gluconacetobacter xylinus (=Acetobacter xylinum) shows variety in acid formation from sugars and sugar-alcohols. Toyosaki et al. proposed new subspecies of G. xylinus (=Acetobacter xylinum) subsp. sucrofermentans in point of acid formation from sucrose and a homology index of 58.2% with the type strain of G. xylinus subsp. xylinus in DNA-DNA hybridization experiments. We tried DNA-DNA hybridization to clarify relationship between acid formation from sugars and classification of G. xylinus. The G + C contents of G. xylinus showed 60.1-62.4 mol% with a range of 2.3 mol%. When type strains of G. xylinus subsp. xylinus, G. xylinus subsp. sucrofermentans, and IFO 3288 forming acid from sucrose, were used as probes, the DNAs from three strains showed 67-100%, 64-89%, and 60-100% similarity to those from sixteen strains including bacteria that form acid from sucrose or not. These results show that homology indexes do not reflect differences of acid formation from sucrose. As a results, the species G. xylinus was proved to be genetically homogeneous.     

Complete genome sequence of NBRC 3288, a unique cellulose-nonproducing strain of Gluconacetobacter xylinus isolated from vinegar

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.     

Taxonomic heterogeneity of strains comprising Gluconacetobacter hansenii

The taxonomic standing of Gluconacetobacter hansenii was clarified through phenotypic characteristics, quinones, DNA base composition, DNA relatedness, and the production of gluconic and ketogluconic acids from glucose. All strains that Gosselé et al. (Syst. Appl. Microbiol., 4, 338-368, 1983) employed in the establishment of Acetobacter hansenii (=G. hansenii) were used in this study. Phenotypic differences were shown among the strains of G. hansenii, suggesting heterogeneity within the species. The major ubiquinone was Q-10 for all strains of G. hansenii, except for strain IFO 3296, which was characterized by Q-9. This excluded IFO 3296 from the species G. hansenii and placed it in the genus Acetobacter. DNA relatedness revealed four distinct homology groups (I, II, III, and IV) among strains of the species. Group I was distinguished from the other genomic groups by a lower G1C range from 58.9 to 59.2 mol%. Groups II, III, and IV showed higher G+C contents of 60.4 to 62.2, 60.8, and 61.7 mol%, respectively. Groups I and IV produced both 2- and 5-ketogluconic acids from glucose, and Group III produced only 2-ketogluconic acid. Group II included strains that produced both 2- and 5-ketogluconic acids and strains that produced only 2-ketogluconic acid. It is clear that G. hansenii consists of genotypically heterogeneous strains comprising four homology groups (I, II, III, and IV). Since group I contains the type strain (IFO 14820(T)=LMG 1527(T)) of the species, this group is designated as the species G. hansenii.     

Influence of nutritional factors on the nature, yield, and composition of exopolysaccharides produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Antagonism among Gluconacetobacter diazotrophicus strains in culture media and in endophytic association

In this study the antagonistic activity among 55 Gluconacetobacter diazotrophicus strains, belonging to 13 electrophoretic types (ETs), in culture media was analyzed. Antagonistic effects were seen only in strains belonging to two ETs named ET-1 and ET-3. Two out of 29 ET-1 strains, and 3 out of 7 ET-3 strains of G. diazotrophicus showed antagonistic effects against many other strains belonging to all the ETs of this species analyzed, and against closely related strains of Gluconacetobacter species, including Gluconacetobacter johannae, Gluconacetobacter azotocaptans and Gluconacetobacter liquefaciens but not against other phylogenetically distant bacterial species. Results showed that the substance responsible of such antagonistic activity is a low molecular mass molecule (approximately 3400 Da), stable from pH 3.5 to 8.5, and very stable at 4 degrees C for 10 months. This substance was sensitive to proteases, and the antagonistic activity was lost after 2 h at 95 degrees C. All of these features show that the substance is related to bacteriocin-like molecules. The antagonistic substance should be chromosomally encoded because ET-3 strains of G. diazotrophicus do not harbor any plasmids. The antagonistic ability of ET-3 strains of G. diazotrophicus could be an advantage for the natural colonization of the sugarcane environment, as was observed in experiments with micropropagated sterile sugarcane plantlets co-inoculated with a bacteriocin-producer strain and a bacteriocin-sensitive strain of G. diazotrophicus. In these experiments, both in the rhizosphere as well as inside the roots, the bacteriocin-sensitive population decreased drastically. In addition, this study shows that inside the plants there may exist antagonistic interactions among endophytic bacteria like to those described among the rhizospheric community.     

Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus.     

Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.     

Genome sequences of the high-acetic acid-resistant bacteria Gluconacetobacter europaeus LMG 18890T and G. europaeus LMG 18494 (reference strains), G. europaeus 5P3, and Gluconacetobacter oboediens 174Bp2 (isolated from vinegar)

Bacteria of the genus Gluconacetobacter are usually involved in the industrial production of vinegars with high acetic acid concentrations. We describe here the genome sequence of three Gluconacetobacter europaeus strains, a very common bacterial species from industrial fermentors, as well as of a Gluconacetobacter oboediens strain.     

Production of 4-keto-D-arabonate by oxidative fermentation with newly isolated Gluconacetobacter liquefaciens

Production of 4-keto-D-arabonate (4KAB) was confirmed in a culture medium of Gluconacetobacter liquefaciens strains, newly isolated from water kefir in Argentina. The strains rapidly oxidized D-glucose, D-gluconate (GA), and 2-keto-D-gluconate (2KGA), and accumulated 2,5-diketo-D-gluconate (25DKA) exclusively before reaching the stationary phase. 25DKA was in turn converted to 4KAB, and 4KAB remained stable in the culture medium. The occurrence of 4KAB was assumed by Ameyama and Kondo about 50 years ago in their study on the carbohydrate metabolism of acetic acid bacteria (Bull. Agr. Chem. Soc. Jpn., 22, 271-272, 380-386 (1958)). This is the first report confirming microbial production of 4KAB.     

DNA base composition of planktonic species of Anabaena (Cyanobacteria) and its taxonomic value

Fifty axenic strains of planktonic Anabaena, including 24 strains of the straight form and 26 strains of the coiled form, were examined for their DNA base composition (GC content). The taxonomic value of their GC content at species level was evaluated by comparing their morphological, physiological and biochemical properties. The DNA base composition determined for all fifty strains ranged from 35.9 to 56.4 mol% GC. The straight-form strains were in the range of 35.9-56.4 mol% GC, while coiled forms were in the range of 38.1-50.3 mol% GC. In general, strains assigned to the same species showed similar DNA base composition. However, of three strains of A. affinis Lemmermann that were separated into two categories, two had 40.6-40.9 mol% GC, and the third strain 45.6 mol% GC. It is noteworthy that the DNA base composition of the newly established species A. eucompacta Li et Watanabe was 45.5 mol% GC, which differed from 39.5 mol% GC of the morphologically close species, A. compacta (Nygarrd) Hickel.     

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524

Aims:  To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced.
Methods and Results:  Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga . xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3·83 and 3·75 g l⁻¹ respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2·5 g l⁻¹. Solid state ¹³C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles.
Conclusions:  The production rate of bacterial cellulose by Ga . xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features.
Significance and Impact of the Study:  Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga . xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.     

Identification of acetic acid bacteria isolated from Indonesian sources,especially of isolates classified in the genus Gluconobacter

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.     

一株高产苯乳酸的古墓土壤葡糖醋杆菌FBFS97的全基因组测序与分析

【背景】苯乳酸(phenyllactic acid,PLA)是一种应用潜力巨大的天然广谱抑菌物质。本课题组前期分离得到一株高产PLA的醋酸菌(acetic acid bacteria,AAB)——葡糖醋杆菌(Gluconacetobacter sp.)FBFS97,但尚未鉴定到种,而且其产PLA的分子机理尚不清楚。【目的】确定FBFS97的种属关系,解析FBFS97的遗传信息,特别是与PLA产生相关的基因。【方法】采用光学显微镜和扫描电镜对FBFS97的菌体形态进行表征,通过16S rRNA基因序列分析对FBFS97进行分类鉴定,并以高效液相色谱分析苯丙氨酸对其产PLA的影响。在此基础上,对FBFS97进行全基因组测序、拼接和基因预测,并进行GO/COG聚类、KEGG代谢通路和VFDB毒力等分析,以及PLA生物合成途径的预测。【结果】根据16S rRNA基因序列的比对结果,结合形态学分析,该菌被鉴定为古墓土壤葡糖醋杆菌(Gluconacetobacter tumulisoli)。将1 000 mg/L苯丙氨酸添加到FBFS97液体培养基中,发酵液中PLA最高浓度可达400 mg/L,为对照组的8倍。该菌的基因组大小为3 988 308 bp,(G+C)mol%含量为66.62%,编码基因3 500个;KEGG代谢通路分析表明,该菌基因组中存在经莽草酸途径合成PLA的所有基因;VFDB毒力预测结果显示,该菌基因组中不存在产生毒素的相关基因。【结论】首次报道了一株高产PLA的AAB——古墓土壤葡糖醋杆菌FBFS97的全基因组序列信息,并发现该菌株的基因组中含有合成PLA的所有相关基因,为后续进一步研究FBFS97产生PLA的生物合成途径提供了理论依据。     

Acetobacter intermedius,sp. nov

Strains of a new species in the genus Acetobacter, for which we propose the name A. intermedius sp. nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia). The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A. europaeus, do, however, not require acetic acid for growth. These characteristics phenotypically position A. intermedius between A. europaeus and A. xylinus, DNA-DNA hybridizations of A. intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A. xylinus, A. aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity. The important features of the new species are described. Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain.     

Isolation and identification of Gluconacetobacter azotocaptans from corn rhizosphere

Six acetic acid producing, diazotrophic bacteria were isolated from soil adhering to corn roots. These isolates were shown to be Gluconacetobacter azotocaptans and they shared some features with G. johannae and G. diazotrophicus but differed on the basis of colony morphology on different media, use of carbon sources and use of l-amino acids as a nitrogen source. The species identity was confirmed using 16S rDNA sequence analysis, PCR amplification of 16S rRNA gene with species-specific primers and amplified rDNA restriction analysis. This is the first report of the presence of this bacteria on corn plants. Scope of the paper: This is the first report of the occurrence and association of Gluconacetobacter azotocaptans with corn.     

Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation

Applied Microbiology and Biotechnology - Oxygen plays a key role during bacterial cellulose (BC) biosynthesis by Gluconacetobacter xylinus. In this study, the Vitreoscilla hemoglobin (VHb)-encoding...     

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

DNA relatedness among bacteriophages of the morphological group C3

Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×10⁶ (C2) to 67 ×10⁶ (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×10⁶. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×10⁶. It is proposed that phage species be defined as phage nucleic acid hybridization groups.     

DNA base composition and DNA relatedness among species of Trichosporon Behrend

The nuclear DNa of 28 species (30 strains investigated) of yeasts classified currently or previously in the genus Trichosporon. was analysed for its molar percentage of guanine + cytosine (mol% G+C). This criterion, together with biochemical characteristics, suggested the separation of the organisms studied into two groups. The first group, which appears related to the Ascomycetes, includes thirteen species with a G+C content lower than 50 mol% (34.7–48.8), and lacks urease (except T. margaritiferum). The second group appears related to the Basidiomycetes and includes fifteen species with a G+C content higher than 50 mol% (57–64) and has the ability to hydrolyse urea.A DNA homology experiment with T. beigelii and twelve other species of the second group showed very low values of complementarity with T. beigelii-labeled DNA. All these species must be considered as taxa other than T. beigelii.