CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide.

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Differential Gene Expression of Brachypodium distachyon Roots Colonized by Gluconacetobacter diazotrophicus and the Role of BdCESA8 in the Colonization.

Alternatives to synthetic nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Nitrogen-fixing bacterium Gluconacetobacter diazotrophicus has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of host plants to G. diazotrophicus colonization is still lacking. In this study, the molecular response of the monocot plant model Brachypodium distachyon was studied during G. diazotrophicus root colonization. The gene expression profiles of B. distachyon root tissues colonized by G. diazotrophicus were generated via next-generation RNA sequencing, and investigated through gene ontology and metabolic pathway analysis. The RNA sequencing results indicated that Brachypodium is actively involved in G. diazotrophicus colonization via cell wall synthesis. Jasmonic acid, ethylene, gibberellin biosynthesis. nitrogen assimilation, and primary and secondary metabolite pathways are also modulated to accommodate and control the extent of G. diazotrophicus colonization. Cellulose synthesis is significantly downregulated during colonization. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) showed decreased cellulose content in xylem and increased resistance to G. diazotrophicus colonization. This result suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The results of this study provide insights for future research in regard to gene manipulation for efficient colonization of nitrogen-fixing bacteria in Brachypodium and monocot crops.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biofabrication of a Hyaluronan/Bacterial Cellulose Composite Nanofibril by Secretion from Engineered Gluconacetobacter.

Naturally occurring polysaccharides, such as cellulose, hemicellulose, and chitin, have roles in plant skeletons and/or related properties in living organisms. Their hierarchically regulated production systems show potential for designing nanocomposite fabrication using engineered microorganisms. This study has demonstrated that genetically engineered Gluconacetobacter hansenii (G. hansenii) individual cells can fabricate naturally composited nanofibrils by simultaneous production of hyaluronan (HA) and bacterial cellulose (BC). The cells were manipulated to contain hyaluronan synthase and UDP-glucose dehydrogenase genes, which are essential for HA biosynthesis. Fluorescence microscopic observations indicated the production of composited nanofibrils and suggested that HA secretion was associated with the cellulose secretory pathway in G. hansenii. The gel-like nanocomposite materials produced by the engineered G. hansenii exhibited superior properties compared with conventional in situ nanocomposites. This genetic engineering approach facilitates the use of G. hansenii for designing integrated cellulose-based nanomaterials.

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Assessment of the cutaneous wound healing efficiency of acidic, neutral and alkaline bacterial cellulose membrane in rat.

Recent research was conducted to evaluate the healing efficiency of bacterial cellulose (BC) as a wound dressing in different pHs and its possibility of being a smart wound dressing that can indicate pHs. BC was produced by environmentally isolated bacterial strains. After washing the best achieved BC, it was floated in normal saline with different pHs with phenol red used as a pH indicator. Finally the wound healing effects of the acidic, neutral and alkaline BC membranes were evaluated in rat cutaneous wounds. Results showed that one of the isolates which its partial 16srRNA genome had 95% similarity with Gluconacetobacter intermedius, had the thickest layer. The microscopic and macroscopic evaluations showed that the acidic BC had the best healing activity. Although the color of the films remained unchanged during the experiments because they were transparent and thin, these changes could not be easily seen. This suggests the use of thicker films such as the ones which are cross linked with some materials (e.g., sterile gauze). In conclusion the pH can affect the healing ability of natural BC and acidic pH had the best wound healing efficiency. In future it is better to use the acidic BC instead of natural one for different wound healing purposes.

Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 µm and a diameter of 10-20 µm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.

Isolation and characterization of active promoters from Gluconacetobacter diazotrophicus strain PAL5 using a promoter-trapping plasmid.

Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through ß-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.

Airborne Bacterial Diversity from the Low Atmosphere of Greater Mexico City.

Greater Mexico City is one of the largest urban centers in the world, with an estimated population by 2010 of more than 20 million inhabitants. In urban areas like this, biological material is present at all atmospheric levels including live bacteria. We sampled the low atmosphere in several surveys at different points by the gravity method on LB and blood agar media during winter, spring, summer, and autumn seasons in the years 2008, 2010, 2011, and 2012. The colonial phenotype on blood agar showed α, ß, and γ hemolytic activities among the live collected bacteria. Genomic DNA was extracted and convenient V3 hypervariable region libraries of 16S rDNA gene were high-throughput sequenced. From the data analysis, Firmicutes, Proteobacteria, and Actinobacteria were the more abundant phyla in all surveys, while the genera from the family Enterobacteriaceae, in addition to Bacillus spp., Pseudomonas spp., Acinetobacter spp., Erwinia spp., Gluconacetobacter spp., Proteus spp., Exiguobacterium spp., and Staphylococcus spp. were also abundant. From this study, we conclude that it is possible to detect live airborne nonspore-forming bacteria in the low atmosphere of GMC, associated to the microbial cloud of its inhabitants.

Tn5 insertion in the tonB gene promoter affects iron-related phenotypes and increases extracellular siderophore levels in Gluconacetobacter diazotrophicus.

TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.

Transfer of Gluconacetobacter kakiaceti, Gluconacetobacter medellinensis and Gluconacetobacter maltaceti to the genus Komagataeibacter as Komagataeibacter kakiaceti comb. nov., Komagataeibacter medellinensis comb. nov. and Komagataeibacter maltaceti comb. nov.

Gluconacetobacter kakiaceti, Gluconacetobacter medellinensis and Gluconacetobacter maltaceti are transferred to the genus Komagataeibacter as Komagataeibacter kakiaceti comb. nov. (type strain, G5-1T=JCM 25156T=NRIC 0798T=LMG 26206T), Komagataeibacter medellinensis comb. nov. (type strain, LMG 1693T=NBRC 3288T=Kondo 51T) and Komagataeibacter maltaceti comb. nov. (type strain, LMG 1529T=NBRC 14815T=NCIMB 8752T).

Gluconacetobacter diazotrophicus PAL5 possesses an active quorum sensing regulatory system.

The endophytic bacterium Gluconacetobacter diazotrophicus colonizes a broad range of host plants. Its plant growth-promoting capability is related to the capacity to perform biological nitrogen fixation, the biosynthesis of siderophores, antimicrobial substances and the solubilization of mineral nutrients. Colonization of and survival in these endophytic niche requires a complex regulatory network. Among these, quorum sensing systems (QS) are signaling mechanisms involved in the control of several genes related to microbial interactions, host colonization and stress survival. G. diazotrophicus PAL5 possesses a QS composed of a luxR and a luxI homolog, and produces eight molecules from the AHL family as QS signals. In this report data are provided showing that glucose concentration modifies the relative levels of these signal molecules. The activity of G. diazotrophicus PAL5 QS is also altered in presence of other carbon sources and under saline stress conditions. Inactivation of the QS system of G. diazotrophicus PAL5 by means of a quorum quenching strategy allowed the identification of extracellular and intracellular proteins under the control of this regulatory mechanism.

Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing.

IMPORTANCE: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis.

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding ß-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.

The bacterial superoxide dismutase and glutathione reductase are crucial for endophytic colonization of rice roots by Gluconacetobacter diazotrophicus PAL5.

Gluconacetobacter diazotrophicus is an aerobic diazotrophic plant-growth-promoting bacterium isolated from different gramineous plants. We showed that reactive oxygen species (ROS) were produced at early stages of rice root colonization, a typical plant defense response against pathogens. The transcription of the pathogen-related-10 gene of the jasmonic acid (JA) pathway but not of the PR-1 gene of the salicylic acid pathway was activated by the endophytic colonization of rice roots by G. diazotrophicus strain PAL5. Quantitative polymerase chain reaction analyses showed that, at early stages of colonization, the bacteria upregulated the transcript levels of ROS-detoxifying genes such as superoxide dismutase (SOD) and glutathione reductase (GR). To proof the role of ROS-scavenging enzymes in the colonization and interaction process, transposon insertion mutants of the SOD and GR genes of strain PAL5 were constructed. The SOD and GR mutants were unable to efficiently colonize the roots, indicated by the decrease of tightly root-associated bacterial cell counts and endophytic colonization and by fluorescence in situ hybridization analysis. Interestingly, the mutants did not induce the PR-10 of the JA-pathway, probably due to the inability of endophytic colonization. Thus, ROS-scavenging enzymes of G. diazotrophicus strain PAL5 play an important role in the endophytic colonization of rice plants.

An efficient method using Gluconacetobacter europaeus to reduce an unfavorable flavor compound, acetoin, in rice vinegar production.

Gluconacetobacter europaeus, one of the microorganisms most commonly used for vinegar production, produces the unfavorable flavor compound acetoin. Since acetoin reduction is important for rice vinegar production, a genetic approach was attempted to reduce acetoin produced by G. europaeus KGMA0119 using specific gene knockout without introducing exogenous antibiotic resistance genes. A uracil-auxotrophic mutant with deletion of the orotate phosphoribosyltransferase gene (pyrE) was first isolated by positive selection using 5-fluoroorotic acid. The pyrE disruptant designated KGMA0704 (ΔpyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and the pyrE gene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption of aldC or als, which encodes α-acetolactate decarboxylase or α-acetolactate synthase, was attempted in KGMA0704. The disruption of these genes was expected to result in a decrease in acetoin levels. A disruption vector harboring the pyrE marker within the targeted gene was constructed for double-crossover recombination. The cells of KGMA0704 were transformed with the exogenous DNA using electroporation, and genotypic analyses of the transformants revealed the unique occurrence of targeted aldC or als gene disruption. The aldC disruptant KGMA4004 and the als disruptant KGMA5315 were cultivated, and the amount of acetoin was monitored. The acetoin level in KGMA4004 culture was significantly reduced to 0.009% (wt/vol) compared with KGMA0119 (0.042% [wt/vol]), whereas that of KGMA5315 was not affected (0.037% [wt/vol]). This indicates that aldC disruption is critical for acetoin reduction. G. europaeus KGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.

Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.

Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose-producing acetic acid bacteria isolated from vinegar.

The phylogenetic position of a cellulose-producing acetic acid bacterium, strain ID13488, isolated from commercially available Colombian homemade fruit vinegar, was investigated. Analyses using nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rRNA gene internal transcribed spacer (ITS) sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated the micro-organism to the genus Gluconacetobacter, and more precisely to the Gluconacetobacter xylinus group. Moreover, the data suggested that the micro-organism belongs to a novel species in this genus, together with LMG 1693(T), a non-cellulose-producing strain isolated from vinegar by Kondo and previously classified as a strain of Gluconacetobacter xylinus. DNA-DNA hybridizations confirmed this finding, revealing a DNA-DNA relatedness value of 81 % between strains ID13488 and LMG 1693(T), and values <70 % between strain LMG 1693(T) and the type strains of the closest phylogenetic neighbours. Additionally, the classification of strains ID13488 and LMG 1693(T) into a single novel species was supported by amplified fragment length polymorphism (AFLP) and (GTG)5-PCR DNA fingerprinting data, as well as by phenotypic data. Strains ID13488 and LMG 1693(T) could be differentiated from closely related species of the genus Gluconacetobacter by their ability to produce 2- and 5-keto-d-gluconic acid from d-glucose, their ability to produce acid from sucrose, but not from 1-propanol, and their ability to grow on 3 % ethanol in the absence of acetic acid and on ethanol, d-ribose, d-xylose, sucrose, sorbitol, d-mannitol and d-gluconate as carbon sources. The DNA G+C content of strains ID13488 and LMG 1693(T) was 58.0 and 60.7 mol%, respectively. The major ubiquinone of LMG 1693(T) was Q-10. Taken together these data indicate that strains ID13488 and LMG 1693(T) represent a novel species of the genus Gluconacetobacter for which the name Gluconacetobacter medellinensis sp. nov. is proposed. The type strain is LMG 1693(T) ( = NBRC 3288(T) = Kondo 51(T)).

Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov., isolated from Takamatsuzuka Tumulus samples before and during the dismantling work in 2007.

Ten strains of Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from the burial mound soil collected before the dismantling and samples collected during the dismantling work on the Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan in 2007. On the basis of the 16S rRNA gene sequence analysis of the isolates, they were accommodated in the genus Gluconacetobacter (class Alphaproteobacteria) and can be separated into four groups within the cluster containing the genus Gluconacetobacter. One of the groups demonstrated a phylogenetic position identical to that of Gluconacetobacter asukensis, which was isolated from small holes on plaster walls of the stone chamber interior of Kitora Tumulus in Asuka village, Nara Prefecture, Japan. The remaining three groups consisted of novel lineages within the genus Gluconacetobacter. A total of four isolates were selected from each group and carefully identified using a polyphasic approach. The isolates were characterized on the basis of their possessing Q-10 as the major ubiquinone system and C18 : 1ω7c (58.5-65.2 %) as the predominant fatty acid. A DNA-DNA hybridization test was used to determine that the three lineages represented novel species, for which the names Gluconacetobacter tumulisoli sp. nov., Gluconacetobacter takamatsuzukensis sp. nov. and Gluconacetobacter aggeris sp. nov. are proposed. The type strains are T611xx-1-4a(T) ( = JCM 19097(T) = NCIMB 14861(T)), T61213-20-1a(T) ( = JCM 19094(T) = NCIMB 14859(T)) and T6203-4-1a(T) ( = JCM 19092(T) = NCIMB 14860(T)), respectively.