key: cord-009862-37ki2pd8 authors: Reis, Veronica Massena; Teixeira, Kátia Regina dos Santos title: Nitrogen fixing bacteria in the family Acetobacteraceae and their role in agriculture date: 2015-03-03 journal: J Basic Microbiol DOI: 10.1002/jobm.201400898 sha: doc_id: 9862 cord_uid: 37ki2pd8 For centuries, the Acetobacteraceae is known as a family that harbors many species of organisms of biotechnological importance for industry. Nonetheless, since 1988 representatives of this family have also been described as nitrogen fixing bacteria able to plant growth promotion by a variety of mechanisms. Nitrogen fixation is a biological process that guarantees that the atmospheric N(2) is incorporated into organic matter by several bacterial groups. Most representatives of this group, also known as diazotrophic, are generally associated with soil rhizosphere of many plants and also establishing a more specific association living inside roots, leaves, and others plants tissues as endophyte. Their roles as plant growth‐promoting microorganisms are generally related to increase in plant biomass, phosphate and other mineral solubilization, and plant pathogen control. Here, we report many of these plant growth‐promoting processes related to nitrogen fixing species already described in Acetobacteraceae family, especially Gluconacetobacter diazotrophicus and their importance to agriculture. In addition, a brief review of the state of art of the phylogenetics, main physiological and biochemical characteristics, molecular and functional genomic data of this group of Acetobacteraceae is presented. Bacteria belonging to the Alphaproteobacteria, order Rhodospirillales, are known for their agricultural applicability. This order is represented by two bacterial families: Rhodospirillaceae and Acetobacteraceae. In general, the etymology of members of the Acetobacteraceae family derives from the Latin acetum or acidum gluconicum þ bacter due to their peculiar main characteristic to produce organic acids during many biotechnological processes, such as vinegar and wine productions. Bacteria of the genus Acetobacter are known since 1898 with the description of A. aceti [1, 2] . However, only at 1988, it means 100 years after the first species description in this family, is that a diazotrophic species was described in this family [3] . At that time, this discovery raised the possibility that bacteria of many other species could also present nitrogen fixing and plant growth promotion properties for agricultural purpose, similar to the rhizobia inoculant for soybeans. This document brings the trajectory of accumulated knowledge about the species Gluconacetobacter diazotrophicus and other diazotrophs genera and species that were described later in this family. The Acetobacteraceae family Bacteria belonging to the Acetobacteraceae family (ex Henrici, 1939) [4] are classified as rods (coccus or ellipsoidal), Gram-negative, mobile, aerobic that conduct an incomplete oxidation of sugars and alcohols to produce organic acids as final product of their metabolism. They have the ability to grow in very acidic environments with pH close to 3.0-3.5, but the optimum range is 5.0-6.5 [5] . They are commonly Peculiarities of some Acetobacteraceae genera Some peculiarities that deviate from the classical description of AAB can be highlighted on the genera and their species described over these last 50 years. The genus Rhodopila was proposed by Imhoff et al. [25] to accommodate a group of purple non-sulfur bacteria that presents vesicular intracytoplasmic membranes, similar to Rhodobacter species, but grows at low pH. The genus Acidiphilium was described after isolation and characterization of heterotrophic, mesophilic bacteria with requirements for high acidity and unable to use elemental sulfur or ferrous [26] . Later on, isolates from acidic hot springs and mine drainage were characterized on the basis of molecular and phenotypic traits, especially related to ion-chelated chlorophyll a type, in the genus Acidisphaera, which differs from other aerobic bacteriochlorophyll-containing (ABC) bacteria by producing zinc-chelated bacteriochlorophyll a (Zn-BChl) [26] [27] [28] . In addition to them, other genera known to present bacteriochlorophyll a type were also phylogenetically clustered into the family Acetobacteraceae, and they are as follows: Roseomonas [7, 29] , Roseococcus [30] , Craurococcus, Paracraurococcus [31] , Rubritepida [32] , Humitalea [33] , and Rhodovastum (proposed but not yet recognized genus) [22] . The genus Acidocella was proposed to accommodate two previously described Acidiphilum species that do not present BChl a and that have been classified as monophyletic unit apart from other Acidiphilium species according to 16S rDNA sequence [34, 35] . The genus Stella is known as a polyprosthecate bacteria characterized by having numerous appendage (from the Greek prostheca) [36] . Although it has being classified in this family, further studies based on polar lipids and 16S rRNA genes of the two type strains species indicated its close relationship to members of Rhodospirillaceae [37] . The characterization of a group of acidophilic methanol-utilizing bacteria leads to reclassification of Acetobacter methanolicus into a new genus, Acidomonas [38] . Afterwards, an emendation of the genus description stated that Acidomonas presents acid tolerance, instead of acidophily [39] . Representatives of the genus Asaia do not grow in presence of methanol, are characterized by poor or nonexistent production of acetic acid from ethanol, and by the absence of growth in presence of 0.35% acetic acid (w/v) [40] . The genera Asaia, Kozakia, Swaminathania, and Neoasaia are related phylogenetically to each other; however, Tsp509I and MboII restriction of the 16S-23S rDNA ITS can differentiate Asaia species from Kozakia and Neosaia-type strains [41] . The genera Muricoccus and Theichococcus were proposed to accommodate isolates that do not produce bacteriochlorophyll a under aerobic conditions and were obtained from building material of a children's day care center, specifically from gypsum liner walls of a children's sleeping room [42] . However, Sanchez-Porro et al. had suggest that T. ludipueritiae and M. roseus, the single species of each genera, should be placed within the genus Roseomonas based on pigment color, carbon metabolism, and fatty acids profiles [29] . The genus Saccharibacter corresponds to isolates able to grow in the presence of high concentrations of glucose (2-40% w/v-optimum at 10% w/v) but that present negligible or very weak productivity of acetic acid from ethanol. The species type strain S. floricola was isolated from pollen collected in Kanagawa, Japan [43] . The genus Acidisoma comprises two species, A. tundrae and A. sibiricum, that were isolated from acidic Sphagnumdominated tundra and Siberian wetlands in Russia [44] . As general characteristic, they are chemoorganotrophic strict aerobes, psychrotolerant, do not possess bacteriochlorophyll a, produce poly-b-hydroxy-butyrate, and are oxidase-and catalase-positive. Representative of the genus Nguyenibacter, N. vanlangensis, was isolated from rhizosphere of rice plants in Vietnam [21] . The main characteristics of this genus are oxidation of acetate to carbon dioxide and water but not lactate, no production of acetic acid from ethanol; growth is weakly positive either on 30% D-glucose (w/v) or in the presence of 0.35% acetic acid (w/v). As isolation procedure, the authors used LGI N-free-medium prior to cultivation onto another medium containing glucose and ethanol as carbon source and pH 3.5 but nitrogen fixation was not assessed. Two genera were proposed during classification of isolates obtained from a flower sample from Thailand, the genus Neokomagatea that comprises N. thailandica and N. tanensis [45] and the genus Swingsia that comprises the species S. samuiensis, but is not formally recognized [24] . Endobacter medicaginis, the first Acetobacteraceae found as legume nodules endophytes, was isolated from surface sterilized nodules of Medicago sativa grown in an acidic soil at the Province of Zamora, Spain [46] . Several phenotypic and genotypic studies served as the basis for reclassification of several species previously described in the Gluconacetobacter genus at the generic level, including nitrogen fixing species [46] [47] [48] [49] [50] . The genus Komagataeibacter were proposed during separation of Gluconacetobacter xylinum group from the Gluconacetobacter liquefaciens group. To date, among all Acetobacteraceae genera only some representatives of the genera Gluconacetobacter, Acetobacter, Komagataeibacter, Swaminathania, Asaia, and Acetobacter are reported as nitrogen fixing bacteria and the strategies used in order to obtain these new species are described in Table 1 [3, 47, [51] [52] [53] [54] [55] 59] . The Gluconacetobacter genus Until late 80s, there was not any report of AAB capable to fix nitrogen but Cavalcante and D€ obereiner [3] isolated on N-free semi-solid media and described the first N 2fixing AAB. They succeed to isolate from sugarcane plants a group of acid-tolerant bacteria able to fix nitrogen even at pH below 3.5 using a minimal medium based on LG medium [64] , named LGI-P medium, that presents 10% of raw sugar as carbon source and pH around 5.5. Afterwards, during phylogenetic study of representatives of the genus Acetobacter, a new genus was proposed by Yamada et al. [18] leading to the elevation of the subgenus Gluconoacetobacter to the generic level. Two species previously classified as Acetobacter were then renamed into this new genus, G. liquefaciens and G. xylinus, based on 16S RNA sequence, predominance of Q-10 quinone type, flagella, pigment production, cellulose production, and fatty acid profile. Later on, during the validation, the name has been corrected to Gluconacetobacter in accordance with Rule 61 of the Bacteriological Code [19] . Since then, a plethora of species isolated from various environments were described into this genus. Nonetheless, after detailed phylogenetic analysis some of them were reclassified into another genus [47, 51, 55, [65] [66] [67] [68] [69] . After 2001, new nitrogen fixing species were described in the genus Gluconacetobacter, G. johannae, and G. azotocaptans [51] . G. swingsii and G. rhaeticus are cellulose-producing acetic acid bacteria isolated from apple juice of fruits cropped in the South Tyrol region of Italy [67] . G. sacchari was isolated from sugarcane and insects [66] . G. saccharivorans and G. nataicola were proposed based on a reclassification study of Gluconacetobacter hansenii strains [70] . G. tumulicola and G. asukensis were isolated from biofilms growing on the surface of the plaster walls of the mural paintings of the Kitora Tumulus in Japan [68] . G. tumulisoli, G. takamatsuzukensis, and G. aggeris were isolated from the burial mound soil collected at Takamatsuzuka Tumulus in Asuka village, Nara Prefecture, Japan [69] . As previously occurred to G. kombuchae, the species G. kakiaceti [62] , G. medellinensis [71] , and G. maltaceti [72] were reclassified into the genus Komagataeibacter [55] based on previous phylogenetic studies using 16S rRNA sequences [47, 50] . The reasons that corroborated the existence of two phylogenetic groups in the genus Gluconacetobacter was discussed by Yamada and Yukphan [13] . According to previous observations, the group 1 included G. liquefaciens, G. diazotrophicus, G. sacchari, G. johannae, and G. azotocaptans, while group 2 included G. xylinus G. hansenii, G. europaeus, G. entanii, G. oboediens, G. intermedius, G swingisii, G rhaeticus, G. saccharivorans, and G. nataicola. Group 1 species differentiate from group 2 by many physiological and morphological traits, such as flagella and motility, water-soluble brown pigment production, production of g-pyrone compounds, and 2,5 di-ketogluconic. Species clustered into these groups also showed other common features such as biotechnological applications and habitats. Ecologically, the species of group 1 were found associated with plants, fruits, or flowers, noteworthy group 2 were generally isolated from fermentation process and processed food, such as vinegar, Kombucha tea, and nata de coco [73] . Nitrogen fixation agents of biocontrol and associated to several plant growth promotion were related to species of group 1, while most species in group 2 were related with industrial applications. Cleenwerck et al. [74] also contributed with genotypic data to reinforce the separation of Gluconacetobacter species at generic level. Recently, G. xylinum group (group 2) were separated from the G. liquefaciens group (group 1) leading to reclassification of several species in the genus Komagataeibacter, including G. kombuchae, a nitrogen fixing species later considered heterotypic synonym of previously named G. hansenii [48, 50] . In the present scenario, the ability to fix nitrogen has being observed in the following Gluconacetobacter species: G. diazotropicus [3, 75] , G. johannae, and G. azotocaptans [51] . The species Gluconacetobacter diazotrophicus (former Acetobacter diazotrophicus) was isolated from roots, stems, and leaves of sugarcane not only in Brazil but also in Argentina, Uruguay, Mexico, Cuba, United States, India, Canada, Egypt, beside others [3, [76] [77] [78] and then from other agricultural crops such as sugar beet, rice, pineapple, coffee, carrot, and many others [79] [80] [81] . It was also isolated from bugs, such as mealybugs commonly found associated to sugarcane crops [82, 83] . This species has been considered as one of the most important diazotrophic bacteria found in high numbers (10 4 -10 6 CFU g À1 plant fresh tissues) and colonizing the inner parts of roots, stems, and leaves of sugarcane [84] . It is a nitrogen-fixing bacterium originally classified as Acetobacter diazotrophicus but later renamed to the genus Gluconacetobacter based on the 16S rDNA sequence and the predominant type of ubiquinone [18, 19] . Physiological characteristics. It is Gram-negative, aerobic but fixes nitrogen in microaerobic conditions. It does not use tricarboxylic acids to grow and is adapted to conditions of high osmolarity and sucrose content (10-30%). In addition, its cultivation in presence of high sugar content revealed that nitrogenase activity is only partially inhibited by the addition of ammonium to the culture medium [85] . It shows high acidity tolerance, fixes nitrogen in presence of nitrate concentrations greater than 10 mM, and reduces the deleterious effect of oxygen concentration to the nitrogenase activity using oxidative metabolism in the periplasmic space at membrane level [3, 86] . Genetic traits. Studies of genetic characterization in G. diazotrophicus started in the early 90s. The first report of the chromosomal localization of nitrogen fixation genes and presence of plasmids in G. diazotrophicus strains was presented during the 6th International Nitrogen fixation with non-legumes in Egypt [87] . Later on, the nif genes, transcription regulatory genes (nifA and ntrBC) and others related to ammonium sensing and transport (glnB, glnD, and amtB) were sequenced and expression of some of them were studied using transcriptional gusA fusion [88] [89] [90] [91] [92] . The nif-fix gene organization found in G. diazotrophicus was similar to that of Azospirillum brasilense, but their products were homologous to those observed to representatives of Rhizobiaceae family and R. capsulatus, considering the GenBank data available at that time [88, 91] . In addition, accessory genes related with nitrogen sensing and metabolism were also studied by sequencing and insertion mutagenesis. At least three copies of genes coding PII-like protein were identified. The copy located upstream to glnA (that codes for glutamine synthetase) was named glnB, because of its homology and conserved organization on many others nitrogen-fixing bacteria. The others PII-like coding genes, glnK1 and glnK2, were located upstream of copies of genes that codes for ammonium or methylammonium transportes, amtB1 and amtB2, respectively [92] . Further characterization of single and double mutants containing gus-fusion suggested that GlnB and GlnK1 are required as positive signals to efficiently relieve repression by GlnK2. In addition, the authors showed that GlnK2 protein clearly has a function different from those of GlnB and GlnK1, since nif gene expression were repressed in glnB glnK1 double mutant under all conditions tested. Based on these studies, the authors suggested that none of the three PII homologs is required for nif gene expression indicating novel regulatory features of G. diazotrophicus PII proteins. The GlnK2 protein acts primarily as an inhibitor of nif gene expression while GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2 [92] . Some years ago a lab consortium, named RIOGENE, announced that the genome of this microorganism was sequenced [60] . Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. The genome size is in the average of others AAB genome already published [93] [94] [95] [96] [97] [98] [99] . Further studies of this microorganism functional genomics have been underway by several researchers group and are leading to a comprehension of the role of its gene contents to its general metabolism, nitrogen fixation, and others plant growth mechanisms [100] [101] [102] [103] [104] [105] [106] [107] [108] . In addition, several mutants have already been obtained and used for functional genomic characterization [100, [102] [103] [104] [105] [106] [107] 109] . The expression of genes related to reactive oxygen species (ROS) detoxification (sod, kat, and gor) was evaluated in G. diazotrophicus grown under nitrogen non-fixing (NFIX) and fixing conditions to elucidate the paradox of oxygen consumption during respiration protection and ROS inhibitory effect on nitrogenase activity and nif gene expression [110] . They observed that growth of this microorganism under nitrogen fixing condition leads to reduction of ROS accumulation and a strong induction of sodA, katE, kat, katC, and gorA genes in comparison to cells grown under non-fixing condition. In addition, sodA and katE gene expression correlates with nifD suggesting that reduction of ROS is essential for the homeostasis during respiratory protection and nitrogen fixation in G. diazotrophicus. Later on, the participation of G. diazotrophicus detoxifying genes during the first step of root colonization was investigated using rice plants [111] . It was shown that ROS accumulates during the first steps of inoculation of G. diazotrophicus wild type and mutant strains as response of a plant defense mechanism. Noteworthy, they found out that GR (glutathione reductase) and SOD (superoxide dismutase) mutants of G. diazotrophicus could not reduce efficiently the ROS accumulated in the period of 1-7 h after inoculation. In addition, they measured PR genes expression and detected that expression of JA/ET pathway gene increased during wild type-plant interaction, but not to both mutants. These data suggest that G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels to circumvent ROS inhibitory effect to nitrogenase activity. Probably this transcriptional regulatory mechanism protects nitrogenase activity during initial stages of plant colonization. Genes homologous to those of alternative asparagine biosynthesis pathway and its role during nitrogen fixation were investigated since free asparagine, as well as others amino acids, inhibits nitrogenase activity [112, 113] . Further, genome analyses revealed that genes of asparaginyl-tRNA and asparagine synthetase orthologs are absent in the G. diazotrophicus genome. However, the correlation between repression of nifD expression and increase of Asparagine level indicated that genes for an alternative route that converts Asp-tRNA Asn into Asn-tRNA Asn by a glutamine-dependent Asp-tRNA Asn amidotransferase B, encoded by the operon gatCAB, might be present. In fact, in G diazotrophicus the presence of gatCAB operon indicates that the ORF GDI2232 encodes an Aspartyl-tRNA synthetase of ND-AspRS type [113] . The role of asparagine and glutamine as N storage molecule in plant tissues, including sugarcane, is a general rule; however, their role in plant bacteria interaction is scarce and requires more investigation. The content of amino acids in sugarcane sap, apoplast, and symplast has been evaluated and shows that Asn is one of the most abundant [114, 115] . It is known that Asn and Gln are amino acids that repress nitrogen fixation in vitro. Noteworthy, the presence of ORFs coding for putative Lasparaginase precursor (GDI3138), L-asparaginase II protein (GDI1250), and ORFs coding putative (aspartate) aminotransferase in G. diazotrophicus genome may represent an adaptive advantage to its endophytic behavior. Nonetheless, a detailed study is necessary to justify this assumption. Kerby and Roberts [116] identified in G. diazotrophicus genome the presence of two ORFs coding predicted proteins similar to R. rubrum CowN (CO weal-nitrogenase) and transcriptional regulator CooA, which belongs to the Crp/Fnr family. The ORFs GDI_3488 and GDI_3487, previously annotated as hypothetical protein [60] , share common features of organization and motifs to cowN and cooA, respectively. Interestingly, the characterization of these genes in R. rubrum using PSI-Blast searches revealed that they are widespread and generally presents similar organization in nitrogenfixing bacteria. The importance of CooA and CowN for Mo-nitrogenase-dependent functioning in the presence of CO was shown to R. rubrum and R. capsulatus, but not to the Fe-dependent nitrogenase of the latter [116, 117] . The authors suggested that CooA and CowN may act as a two component system that senses and modulates the Modependent nitrogenase activity by protecting it in the presence of CO in R. capsulatus and also in many others nitrogen fixing organisms. However, how this protection mechanism works and its relevance to nitrogen fixation is not yet understood. Here, we presented some examples of the functional genomics studies that have already been published about G. diazotrophicus, but many others have already been thoroughly reviewed or are underway [118, 119] . Colonization. It is considered an endophytic bacterium because it has low rate of survival in the soil and was found colonizing the intercellular space of plant tissues of sugarcane [76, 77, 120, 121] . This bacterium is located in different parts of the plant as described by Reis et al. [79] and James et al. [120] . They demonstrated during "in vitro" inoculation studies under controlled conditions, that G. diazotrophicus enters into sugarcane micropropagated plants through the tissue of secondary roots then the bacteria penetrated inner tissues and colonize the intercellular spaces (apoplast). Other possible points of infection are wounds and the stomata of sugarcane plants [121] . It also colonizes tip of roots and root hairs of other plants such as wheat, sorghum, and rice as showed using reporter genes [123, 124] . At field conditions, the main route of transmission is by vegetative multiplication of stem pieces of sugarcane, although the trash should also serve as an alternative inoculum source when incorporated into the soil [79] . Another possibility to introduce this bacterium in plants appears to be related to the phloem sap sucking by the insects (mealybugs) presenting this species in the lymph and living within sugarcane leaves sheath pocket [82] . Caballero-Mellado and Mart ınez-Romero [83] hypothesized that these insects and also micorrhyzal spores could be responsible for local dispersion of this species within short distance while sugarcane setts and bud chips used to propagate sugarcane could carry the bacteria to further distant geographic regions. No further evidence that these insects are responsible for the dispersal of G. diazotrophicus species is reported, although it is plausible that this occurs. Unfortunately, ecological studies are underemphasized nowadays and this data is not available to a great number of newer described species. Oliveira et al. [125] observed colonization of micropropagated sugarcane using this species in combination with four other strains of diazotrophs. Promising results were obtained when micropropagated sugarcane plants were inoculated with the type strain PAL5 in combination with small doses of nitrogen as shown by Moraes and Tauk-Tornisielo [126] . Oliveira et al. [125] showed that the combined inoculation of five endophytic diazotrophs promotes a synergistic effect when compared with the individual bacterial inoculation in micro-propagated plants of sugarcane in pots and later at field conditions where increases of up to 30% in the accumulation of N via BNF were observed in two varieties of sugarcane planted in three soil types [127] . Plant growth-promoting strategies. Sevilla et al. [128] described the contribution of inoculation with G. diazotrophicus in the nutrition of sugarcane and found that other factors influence on plant growth, such as growth regulators production. The ability of G. diazotrophicus to fix nitrogen and growth promotion of sugarcane was evaluated by comparing plants inoculated with the wild type (PAL5 T ) and an Nif mutant (MAd3Acarry a nifD mutation) in two experiments [128] . Both, the type strain and the mutant, colonized sugarcane plants and persisted in mature plants. Under conditions of nitrogen deficiency, plants inoculated with PAL5 T generally grew better and had a higher content of total nitrogen 60 days after planting when compared to plant inoculated with the Nif mutant. These results indicate that the transfer of fixed nitrogen from G. diazotrophicus to sugarcane may be an important mechanism for the growth promotion in this association. When nitrogen was not limiting, the stimulation of growth was also observed in plants inoculated with both bacteria suggesting an additional effect of G. diazotrophicus inoculation related to growth promotion [128] . This contribution to growth was also observed by Riggis et al. [129] ] G. diazotrophicus was inoculated into maize plants. Among plant-growth substances produced by G. diazotrophicus, the indole acetic acid and gibberellins A1 and A3 are phytohormones that act on plant root growth and development of aerial part tissues [130, 131] . G. diazotrophicus synthetize gluconic acid by the extracellular oxidation of the glucose by the action of the enzyme glucose dehydrogenase (GDH-PQQ), localized in the perisplasmic space [86, 132] , leading to the production of gluconic acid. This mild non-corrosive acid can, besides lowering the pH, promote chelation and exchange reactions and has been associated with phosphate and zinc solubilisation/chelation by G. diazotrophicus [133] [134] [135] [136] [137] [138] . Saravanan et al. [139] observed that G. diazotrophicus grown in Zn-amended broth suffers deformation leading to pleomorphic, aggregate-like cells. Noteworthy, characterization of a mutant unable to grown in the presence of Zn, Co, and Cd salts revealed that the product of czcA gene is responsible for G. diazotrophicus resistance to these heavy metals [140] . Biological control. Another promising effect of G. diazotrophicus inoculation is related to the biological control of other microrganisms, such as Xanthomonas albilineans [141, 142] , Colletotrichum falcatum [143] , Helminthosporium spp. [144] , and Fusarium spp. [145] . By cDNA-AFLP analysis, some plant genes (using leaf tissue) involved in biocontrol activity was identified [108] . These results indicate that inoculation stimulates genes involved in plant defense such as genes controlling the ethylene defense pathway. This pathway is activated when sugarcane micropropagated plants are inoculated with endophytic bacteria [146] [147] [148] . Even nematodes can be controlled by inoculating G. diazotrophicus as demonstrated by Chawla et al. [149] that used the isolate number 35-47 to control Meloidogyne incognita in cotton. In contrast to G. diazotrophicus, which inhabits inner plant tissues as an endophyte, G. johannae and G. azotocaptans were only found colonizing the rhizosphere of coffee plants [51] . Later on, G. azotocaptans was also isolated from the rhizosphere of corn [144] . Little information is available about other AAB-N 2fixing plant interaction. In the case of G. azotocaptans, Mehnaz and Lazarovits [144] conducted a trial inoculating plants of four varieties of maize in a greenhouse experiment using sterile soil substrate. The inoculation consisted of G. azotocaptans, Azospirillum lipoferum, and Pseudomonas putida. At 30 days after planting, the authors observed greater root growth and dry mass of the aerial part of the inoculated pots and also observed that some of the strains isolated from the rhizosphere of maize in Canada presented significant plant growth expressed as increased root/shoot mass compared with non-inoculated plants in sand and/or soil, depending on the combination of bacteria and maize variety tested. The genus Komagataeibacter was proposed to group members of Gluconacetobacter species that cluster closely to G. xylinus [150] . Most of representatives of this new genus are known to be of industrial application but two of them are also nitrogen-fixing bacteria. The species Gluconacetobacter kombuchae considered a heterotypic synonym of previously named G. hansenii [48] was lately reclassified as Kamagataeibacter hansenii. It was isolated during a survey of bacteria associated to Kombucha tea together with Acetobacter nitrogenifigens [52] . Kombucha tea is a fermented beverage that contains an association of yeast and bacteria that takes 7-10 days to be prepared. The authors utilized aliquots of the final preparation of the tea to inoculate solid plates containing LGI medium described by Cavalcante and D€ obereiner [3] but with final pH 4.5 for its isolation. This bacterium also grows in the presence of 30% of glucose or sucrose and can produce cellulose. Sequences deposited at GenBank and described as partial nifA (EF620555) and nifH (DQ141200) coding regions do not share identities/similarities to deduced amino acids of others nitrogen fixing bacteria, as indicated by blast analysis. Komagataeibacter (Gluconacetobacter) kakiaceti was isolated by Iino et al. [62] from traditional kaki vinegar (produced from fruits of kaki, Diospyros kaki Thunb). Recently, the genome of the K. kakiaceti JCM 25156 was sequenced and revealed presence of genes homologous to nif and other regulatory proteins related to Nmetabolism. Its whole genome sequence (WGS) is available at GenBank (scaffolds accession number NZ_BAIO01000001-NZ_BAIO01000947). However, up to date no further experimental evidence of N-fixation by this species was reported. Swaminathania salitolerans was classified as a new genus and new species by Loganathan and Nair [53] . These authors identified new isolates tolerant to salinity stress using rhizosphere, roots, and stems of mangroveassociated wild rice plants (Porteresia coarctata Tateoka). The medium used to obtain these new isolates was a semisolid LGI culture medium without the addition of nitrogen, final pH 5.5 with the addition of 250 mM NaCl. Samples were collected in the city of Tamil Nadu in India, where 41 isolates were obtained and identified as rods, Gram-negative, mobile, and with peritrichous flagella. Strains grew well in the presence of increasing concentrations of acetic acid (0-35%) in a very acid pH, 3.5 and also could grow in the presence of 3% NaCl and 1% KNO 3 . Isolates were able to fix nitrogen and solubilized phosphate in the presence of this level of salt, mimicking the location of its isolation. The colonies grown on LGI medium are initially yellow orange but become darker after aging, smooth, and raised margin, characteristics commonly found in this bacterial family. In this case, there is a description of fixing species using Acetylene Reduction Assay (ARA) to estimate nitrogenase activity during growth in semisolid LGI medium, besides PCR amplification of nifD. But no further works using this bacterial species were published and therefore its agricultural importance is unknown. The Asaia genus was first described with a single species A. bogorensis and then six more species were included: A. siamensis, A. krungthepensis, A. lannaensis, A. platycodi, A. prunellae, and A. astilbes [40, 61, 151, 152] . These strains produce low quantities of acetic acid from ethanol and grew in medium with dulcitol as the sole carbon source, indicating that they belong to the genus Asaia [54] . The isolates also grow on LGI containing 10% sucrose as carbon source (LGI-P), but in this case it is acidified to pH 5.0 with acetic acid. Interestingly, this genus includes species described from samples taken from the interior of insects as mosquito Anopleles and Plasmodium that are vectors of malaria and dengue fever and cause of its sanitary importance, many studies describe the presence of various species of mosquitoes in association with this group of bacteria [153] . In India, a study was performed in order to isolate diazotrophs from three different samples: one flower called Michalia champaca, from the Anopheles mosquitoes, and from ants, such as in Tetraponera rufonigra, Pseudomyrmex, Cephalotes, and Paraponera [54, 154] . It is important to emphasize that these isolates where obtained using N-free LGI medium as described by Magalhães et al. [155] to isolate Azospirillum amazonense modifying the final pH of the medium from 6.0 to 4.5 replacing sulfuric acid by acetic acid. The same medium was used to characterize the G. diazotrophicus species in 1988 [3] . Samaddar et al. [54] isolated Asaia spp. as endophytes based on the surface disinfection of the plant tissue and confirmed their ability to fix nitrogen by using ARA to estimate nitrogenase activity, amplification, and sequencing of nifH-like gene. Asaia bogorensis (MTCC 4041 T ), A. siamensis (MTCC 4042 T ), and A. platycodi AS6 strains were positive to these tests. They formed pink colonies, shiny, smooth, with entire margin in agar plates containing AG medium composed of D-glucose (0.1%), glicerol (1.5%), peptona (0.5%), yeast extract (0.5%), malt extract (0.2%), CaCO 3 (0.7%), and agar (1.5%) as described by Yamada et al. [40] . This pigmentation increased after prolonged incubation at 4°C. All of these isolates were classified as aerobic grow at pH 4.5 and 30°C. Acetate and lactate are oxidized to carbon dioxide and water but the activity was considered low. Partial sequences of nifH-like genes from several isolates and Asaia type strains were obtained and, as well as their genomes [63] , are deposited at GenBank [54] . Noteworthy, up to date blast analysis revealed that these sequences blast only among them and with several partial nifH-like sequences from unculturable clone or chlorophyllide reductase subunit X (bchX) partial gene sequences from several microbial genome or unculturable clones (Fig. 1) . Although this genus is the oldest of Acetobacteraceae family, the description of diazotrophs representatives was first raised by the description of Acetobacter diazotrophicus in the 80s. However, based on detailed taxonomic and phylogenetic studies, this species was reclassified and renamed into the genus Gluconacetobacter. No other species of the genus Acetobacter had been described as diazotrophic until 2005 when Muthukumarasamy et al. [57] presented various isolates belonging to A. peroxydans that fix nitrogen. Most of the isolates were obtained from samples of flooded rice cultivated in India but studies of nifH amplification and ARA confirmed that even in the type strain of A. peroxydans LMG 1635 T these characteristics were present [57] . Shortly thereafter, another study presented the description of the second species of nitrogen-fixing Acetobacter named A. nitrogenifigens based on isolates obtained from Kombucha tea in India [52] . The nitrogen fixing Acetobacter species A. nitrogenifigens shows polar flagella similar to those of Gluconobacter [52] . It also produces brown pigment and g-pyrone compounds, suggesting that 2-ketogluconate and 2,5-diketogluconate are also produced as found in Gluconobacter and Gluconacetobacter [18, 86, 157] . Although claimed as positive for ARA, the A. nitrogenifigens RG1 partial nifH sequence deposited at GenBank (AY952470) do not blast with any nifH coding protein as also observed to K. hansenii (RG3). An overview of the source of isolation and data about nitrogenase activity and nif genes to all these species are shown in Table 2 . Figure 1 . Maximum Likelihood tree of partial NifH and BchX proteins selected from protein sequences blast analysis using nifH gene deduced protein from G. diazotrophicus and Asaia bogorensis as query. Maximum Likelihood method was based on the Dayhoff matrix model in MEGA5 [156] . The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrap values). The agricultural application of species and strains belonging to Acetobacteraceae family will be based almost entirely in a single species, G. diazotrophicus. This species is the oldest described and characterization of its agricultural potential to important crops like sugarcane was quite widespread in Brazil and other countries. For the other nitrogen fixing species descriptions of use are rare or no report of agricultural application is available. The use of diazotrophs in agriculture has been explored for over 30 years and its apex in the past century, the decade of 80-90, then the description of G. diazotrophicus. One of the first reports that populations of diazotrophs could be affected by increasing doses of N-fertilizer was made by Vose et al. [158] in sugarcane which showed that high levels of mineral N caused a significant reduction in the acetylene reduction activity, very popular method which measures the indirect activity of the nitrogenase enzyme acting as a competitive inhibitor. This effect was believed to inhibit this enzyme synthesis. In 1993, after the description of G. diazotrophicus, studies conducted in Mexico by Fuentes-Ram ırez et al. [159] reported that the association between G. diazotrophicus and sugarcane could be severely limited by high N-fertilization, which would explain the decrease in acetylene reduction activity. In their study, the crops fertilized with 120 kg N ha À1 showed higher number of isolates than the plots fertilized with 300 kg N ha À1 , levels not applied in Brazil. Muthukumarasamy et al. [160, 161] obtained similar results in India for G. diazotrophicus. They suggest that this effect was not directly related to the presence of high levels of nitrogen fertilizer in sugarcane crop since this bacterium is able to grow and fix nitrogen "in vitro" in presence of high concentrations of NO 3 (60 mM). It is more likely that at these high N doses the physiological state of the plant undergoes changes and subsequently influences negatively the population of this organism. Muñoz-Rojas and Caballero-Mellado [162] observed a negative effect on G. diazotrophicus population in the presence of high doses of nitrogen appled in sugarcane planted in Mexico. These results were confirmed by Reis Jr. et al. [163] using two sugarcane varieties planted in a sand soil fertilized with 300 kg de N ha À1 in comparison with the control without N application. Only the variety SP792312 presented plant with high levels of total N in the fertilized plots and lower numbers of G. diazotrophicus. Medeiros et al. [164] utilized different sources of nitrogen and observed that G. diazotrophicus reduced acetylene reduction activity in the presence of high levels of N. Studies conducted in India with the application of G. diazotrophicus were repeatedly evaluated by Suman et al. [153] [154] [155] . They reported that the population of G. diazotrophicus was influenced by increased doses of N-fertilizer and that N efficiency in sugarcane increased in the presence of G. diazotrophicus inoculation in greenhouse experiments [166] . Later on, Suman et al. utilized one strain of G. diazotrophicus, named IS100, besides strains of A. brasilense and Azotobacter chrococcum to evaluate nitrogen efficiency applied in increased doses on sugarcane planted in India [167] . G. diazotrophicus showed the best results of crop yield, followed by its combination with A. chrococcum and A. brasilense. Application of G. diazotrophicus was also evaluated in the germination of stem pieces of sugarcane by De la Cruz et al. [168] in Philippines. These authors tested inoculation with different cell densities (10 8 , 10 10 , and 10 12 cells ml À1 ) and methods of application (spray, immersion for 2 h and dipping during 2 min). They observed that inoculation led to increase in percentage survival plant height and shoot/root biomass when compared to the control at 45 days after planting. Introduction of microbial inoculant in 10 12 ml À1 cells by immersion method produced taller plants with greater biomass and root compared to other treatments and uninoculated control. Strains of Gluconacetobacter diazotrophicus, Azospirillum amazonense, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, and Burkholderia tropica species were applied in sugarcane using pots filled with 60 kg of soil and also field experiments planted in three different soil types in São Paulo and Rio de Janeiro states of Brazil showing contributions of the biological process with higher crop yields of different varieties SP70-1143, SP81-3250, RB72454, RB867515 [125, 127, 169, 170] . Oliveira et al. [127] used the technique of d 15 N (natural abundance of 15 N in the soil) and tested seven types of inoculants and found that the inoculant containing five strains described above showed the best results. These authors also quantitated the contribution of BNF showing that the mixture of five strains obtained 29.2% of the accumulated N derived from the air. Schultz et al. [170] utilized the same five strains and modifications of the d 15 N method of BNF quantification of soil applied in the sugarcane yield of RB72454 and RB867515 varieties and showed that plant biomass increased, but found no contributions of nitrogen fixation process by the inoculation. In order to understant how the sugarcane was colonized by this mixture, a fluorescent in situ hybridization (FISH) analysis based on rRNA-targeted oligonucleotide probes confirmed that in micropropagated sugarcane inoculated with this mixture of five species reached the endophytic habitat of micropropagated sugarcane plantlets through active infection of the root cap and emerging zone of secondary roots, although with different efficiencies due to apparently different competitiveness for colonization [133] . Maheshkumar et al. [133] observed that this species was able to solubilize rock phosphate "in vitro," and it could be one of several effects that can promote plant growth after inoculation. Sugar beet has also been used to check the response of G. diazotrophicus inoculation as described by Jambukar and Wange [155] . In 2009, Tian et al. [171] observed the effect of G. diazotrophicus inoculation in different maize genotypes, 17 hybrids, and 10 sweet corn varieties planted in Canada. Colonization of 11 hybrids and 9 sweet corn varieties by G. diazotrophicus was confirmed using species specific primers, but populations were quantified only in the order of 200-3000 cells g À1 of plant tissue. G. diazotrophicus is known worldwide for nitrogen fixation but this is only one of its mechanisms of interest for agriculture and other industrial processes. For example, G. diazotrophicus strain SRT4 has genes for the production of levan-sucrase both endo and exo levanases which are expressed under stress conditions [172, 173] . Another product of its growth is bacteriocins that may act to control growth of other microorganisms or even other strains of the same species [174] . This bacteriocin is constitutively expressed in different conditions of culture medium and dependent on the strain tested. However, is there G. diazotrophicus as commercial product for use in agriculture? The answer is yes. Descriptions of products containing G. diazotrophicus can be found elsewhere. In Argentina, the ene-2 Endophyte-Plus 1 sold by the company ARBO SRL Laboratory, is recommended to be applied as an inoculant for wheat, maize, soybean, and tomato (http://www.arbolab.com.ar/ es/productos/2lvl/prom.html). In Mexico, the company Agro Organics GAIA sells a product containing a mixture of G. diazotrophicus and the fungi Penicillium called Glubac 1 (http://www.organicosgaia. com.mx/biotransferentes-de-nutrientes.html). In Brazil, origin of the G. diazotrophicus description, a patent based on a microorganism species or strain is not allowed, but to several other countries it is. In the United States, there is a patent for use of several nitrogen fixing bacteria, including G. diazotophicus, to enhance plant growth in cereals (US 7393678 B2), and other claiming its use to reduce N fertilization in sugar rich plants, especially sugar beet (20110225679), showing that it can be part of a product for agricultural use. In Brazil, bioprocess can be a matter of patent claims, such as the growth conditions of this bacterial to the production of biomass and fermentation products (PI0917666-7 A2). However, a good product for the industry needs to possess a long shelf-life in order to reduce the costs and facilitates the distribution. Unfortunately, a few studies have developed vehicles and protective substances that increase the longevity of cells of this species. Nita et al. [175] tested several substances such as cell protective for G. diazotrophicus under different temperatures (4 and 25°C). Efficacy was evaluated in tests of wheat inoculation under greenhouse and field conditions. The best method tested was the application of molasses (cane syrup) with 0.1% (w/v) of NH 4 Cl. Trehalose, Arabic gum, and Polyethylene glycol 300 (PEG 300) presented the best results. Addition of L-ascorbic acid (0.02% w/v) to the preservation medium also enhanced the efficacy of the substances used as protectors. After 8-9 months of stock ay 4°C, G. diazotrophicus (strain L1) showed the best results of shelflife in the presence of arabic gum (5% w/v) and PEG 300 (5% w/v), respectively, and also keeping the growth promotion effect. Silva et al. [176] tested a polymer based on carboxymethylcellulose on the survival of G. diazotrophicus strain PAL5 T with a shelf life of 10 9 cell ml À1 for 120 days. To date, data about agricultural application of other nitrogen fixing Acetobacteraceae are restricted to Asaia and it is based on a single report of Weber et al. in 2010 [177] that utilized a single strain of A. bogorensis (AB 219) as inoculant for pineapple and monitored colonization by using agar plates of JNFb medium (malate as a carbon source and final pH 5.8) and population by the Most Probable Number (NMP). The growth and fruiting of pineapple were benefited from the inoculation of A. bogorensis (strain 219) associated with irrigation and increasing doses of organic fertilizer (compost). Since the discovery of Gluconacetobacter diazotrophicus in 1988, many other diazotrophs belonging to the family Acetobacteraceae were described as nitrogen fixing species, but this number can increase. The strategy to isolate and identify new species generally is not based on criteria of biological nitrogen fixation ability and this character is not a discriminatory one. Interestingly, we observed that some researcher groups used the nitrogen free LGI medium as a strategy to obtain new isolates. It is expected that using N-free medium during isolation process can enrich populations of nitrogen fixing bacteria leading to recovery of many of them from environmental samples. In addition, the original pH of LGI was 5.5, but several new species were described with a simple modification of the final pH to levels lower than 5.0. Since representatives of this family are adapted to acidic environment, lowering pH can be considered another strategy used by many authors to isolate and describe new species of nitrogen fixing Acetobacteraceae. It is noteworthy that as many as new species have been described over the last 25 years, publications containing studies of their ecology and distribution diminished considerably. G. diazotrophicus is the most studied nitrogen fixing bacteria of this family for agricultural application. Since the beginning, several studies describing its survival, habitats, mode of plant colonization, and transference to new hosts have accumulated in the literature. Based on them, the description of G. diazotrophicus as a true endophyte was proposed and accepted. The endophytic behavior of G. diazotrophicus is based on many ecological surveys and studies while these data are lacking to other species described. Actually, nowadays publications of ecological research are an exception when we compare with the increasing numbers of species description mainly based on a set of physiological and molecular data, small numbers of specimens or even only one representative. Nitrogen fixation is a biological processes well characterized and understood, at to some points, in pure culture and in vitro that occurs when an appropriate energy source is available in combination with the optimal temperature, pH and controlled O 2 concentration. Nonetheless, it is not an easy task to really prove that a single strain is responsible for part of the assimilated nitrogen in plant, especially under field conditions. In general, the main effects that are easily identified in plants that establish association with non-symbiotic nitrogen fixing bacteria are root surface enhancement, increased grain production, and early maturation. Besides nitrogen fixation ability, G. diazotrophicus also produces growth hormones such as auxins and gibberellins and also can be considered PGPB when compared to A. brasilense. In agricultural perspective the application of Acetobacteraceae species can contribute to plant growth and improve nitrogen assimilation of the host plant. However, under field conditions the effect of the number of bacteria in the plant versus the contribution of biological nitrogen fixation and/or plant growth promotion is not clearly established. It is already reported that introduced populations can undergo changes not only on their physiological aspects, but also in genomic aspects. So far, over more than 30 years of studies most of the knowledge of these aspects of the bacterium-plant interaction is based on analysis conducted under controlled laboratory conditions. Although the centennial knowledge of this versatile bacterial family to industrial application, a lot has to be done about the potential of the nitrogen fixing Acetobacteracea to agricultural application and even to many other industrial biotechnological processes is limited yet. To improve agricultural use or even to broaden the industrial purpose of these nitrogen fixing Acetobacteraceae species depends on development of new biotechnological data. For development of new biotechnological application and products, it will be necessary to increase knowledge and exploit the genomic potential for adaptation, competition, and survival of these bacteria. Introdution of certain species to different plants and/or environmental conditions has to be explored. Besides, the demand of development of methods, easily applied under field conditions, for bacterial inoculation, monitoration, and validation must be constantly considered. In addition, further efforts to characterize the ecology related to these microorganisms and plant relationship is essential. Selection or development of genetically modified bacteria adapted to field competition, stress, and interaction with other components of the microbiota will be one of the goals to improve the inoculation technology worldwide. 1864. M emoire sur la fermentation ac etique € Uber die Arten der Essigbakterien A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane Intra-and intergeneric similarities of the ribosomal ribonucleic acid cistrons of Acetobacter and Gluconobacter The family Acetobacteraceae: the Genera Acetobacter Biotechnological applications of acetic acid bacteria Roseomonas, a new genus associated with bacteremia and other human infections A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease Granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae Comparison of two bacteremic Asaia bogorensis isolates from Europe Acetobacter cibinongensis bacteremia in human Acetobacter indonesiensis pneumonia after lung transplant Genera and species in acetic acid bacteria Taxonomic studies on acetic acid bacteria and allied oxidative bacteria isolated from fruits. A new classification of the oxidative bacteria The flagellation and taxonomy of genera Gluconobacter and Acetobacter with reference to the existence of intermediate strains Essai sur la systematique des acetobacters Gluconoacetobacter, a new subgenus comprising the acetate-oxidizing acetic acid bacteria with ubiquinone-10 in the genus Acetobacter The phylogeny of acetic acid bacteria based on the partial sequences of 16S ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to the generic level Validation list no. 64: validation of publication of new names and new combinations previously effectively published outside the IJSB LPSN-list of prokaryotic names with standing in nomenclature Nguyenibacter vanlangensis gen. nov., sp. nov., an unusual acetic acid bacterium in the a-Proteobacteria Rhodovastum atsumiense gen. nov., sp. nov., a phototrophic alphaproteobacterium isolated from paddy soil Sediminicoccus rosea gen. nov., sp. nov., isolated from the sediment of a eutrophic lake Swingsia samuiensis gen. nov., sp. nov., an osmotolerant acetic acid bacterium in the a-Proteobacteria Rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria Acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments Acidisphaera rubrifaciens gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from acidic environments Bergey's Manual 1 of Systematic Bacteriology: Volume Two: The Proteobacteria, Part A: Introductory Essays Transfer of Teichococcus ludipueritiae and Muricoccus roseus to the genus Roseomonas, as Roseomonas ludipueritiae comb. nov. and Roseomonas rosea comb. nov., respectively, and emended description of the genus Roseomonas Phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of Roseococcus thiosulfatophilus gen Proposal of Craurococcus roseus gen. nov., sp. nov. and Paracraurococcus ruber gen. nov., sp. nov., novel aerobic bacteriochlorophyll a-containing bacteria from soil Rubritepida flocculans gen. nov., sp. nov., a new slightly thermophilic member of the a-1 Subclass of the Proteobacteria Humitalea rosea gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium of the family Acetobacteraceae isolated from soil Acidiphilium aminolytica sp. nov.: an acidophilic chemoorganotrophic bacterium isolated from acidic mineral environment Transfer of Acidiphilium facilis and Acidiphilium aminolytica to the genus Acidocella gen. nov., and emendation of the genus Acidiphilium Stella, a new genus of soil prosthecobacteria, with proposals for Stella humosa sp. nov. and Stella vacuolata sp. nov Phylogenetic relationships of the genera Stella, Labrys and Angulomicrobium within the "Alphaproteobacteria" and description of Angulomicrobium amanitiforme sp. nov Acidomonas gen. nov., incorporating Acetobacter methanolicus as Acidomonas methanolica comb. nov Emendation of the genus Acidomonas Urakami, Tamaoka, Suzuki and Komagata 1989 Asaia bogorensis gen. nov., sp. nov., an unusual acetic acid bacterium in the alpha-Proteobacteria Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the a-Proteobacteria Teichococcus ludipueritiae gen. nov. sp. nov., and Muricoccus roseus gen. nov. sp. nov. representing two new genera of the a-1 subclass of the Proteobacteria Saccharibacter floricola gen. nov., sp. nov., a novel osmophilic acetic acid bacterium isolated from pollen Acidisoma tundrae gen. nov., sp. nov. and Acidisoma sibiricum sp. nov., two acidophilic, psychrotolerant members of the Alphaproteobacteria from acidic northern wetlands Neokomagataea gen. nov., with descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., osmotolerant acetic acid bacteria of the a-Proteobacteria Endobacter medicaginis gen. nov., sp. nov., isolated from alfalfa nodules in an acidic soil Nitrogen-fixing and cellulose-producing Gluconacetobacter kombuchae sp. nov., isolated from Kombucha tea Differentiation of species of the family Acetobacteraceae by AFLP DNA fingerprinting: Gluconacetobacter kombuchae is a later heterotypic synonym of Gluconacetobacter hansenii Subdivision of the genus Gluconacetobacter Yamada, Hoshino and Ishikawa 1998: the proposal of Komagatabacter gen. nov Description of Komagataeibacter gen. nov., with proposals of new combinations (Acetobacteraceae) Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants Novel nitrogen-fixing Acetobacter nitrogenifigens sp. nov., isolated from Kombucha tea Swaminathania salitolerans gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (Porteresia coarctata Tateoka) Nitrogen fixation in Asaia sp. (family Acetobacteraceae) 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 Diversity of acetic acid bacteria in Indonesia, Thailand, and the Philippines Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice Asaia siamensis sp. nov., an acetic acid bacterium in the alpha-proteobacteria Isolation and nitrogen fixing efficiency of a novel endophytic diazotroph Gluconacetobacter diazotrophicus associated with Saccharum officinarum from southern districts of Tamilnadu Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus PAL5 Asaia astilbes sp. nov., Asaia platycodi sp. nov., and Asaia prunellae sp. nov., novel acetic acid bacteria isolated from flowers in Japan Gluconacetobacter kakiaceti sp. nov., an acetic acid bacterium isolated from a traditional Japanese fruit vinegar Draft genome sequence of Asaia sp. strain SF2. 1, an important member of the microbiome of Anopheles mosquitoes Soil bacteriological studies. Further contributions to the physiology and morphology of the members of the Azotobacter group Acetobacter europaeus sp. nov., a main component of industrial vinegar fermenters in Central Europe Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug Description of Gluconacetobacter swingsii sp. nov. and Gluconacetobacter rhaeticus sp. nov., isolated from Italian apple fruit Gluconacetobacter tumulicola sp. nov. and Gluconacetobacter asukensis sp. nov., isolated from the stone chamber interior of the Kitora Tumulus 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 Reclassification of Gluconacetobacter hansenii strains and proposals of Gluconacetobacter saccharivorans sp. nov. and Gluconacetobacter nataicola sp. nov Gluconacetobacter medellinensis sp. nov., cellulose-and non-cellulose-producing acetic acid bacteria isolated from vinegar Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium Genera and species in acetic acid bacteria Phylogeny and differentiation of species of the genus Gluconacetobacter and related taxa based on multilocus sequence analyses of housekeeping genes and reclassification of Acetobacter xylinus subsp. sucrofermentans as Gluconacetobacter sucrofermentans (Toyosaki et al. 1996) sp. nov. , comb. nov Acetobacter diazotrophicus sp. nov., a nitrogen-fixing acetic acid bacterium associated with sugarcane A nitrogen-fixing endophyte of sugarcane stems (a new role for the apoplast) Recent advances in BNF with non-legume plants Gluconacetobacter diazotrophicus: a natural endophytic diazotroph of Nile Delta sugarcane capable of establishing an endophytic association with wheat Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat Coffea arabica L., a new host plant for Acetobacter diazotrophicus, and isolation of other nitrogen-fixing acetobacteria Natural endophytic occurrence of Acetobacter diazotrophicus in pineapple plants Acetic acid bacterial biota of the pink sugar cane mealybug, Saccharococcus sacchari, and its environs Limited genetic diversity in the endophytic sugarcane bacterium Acetobacter diazotrophicus Technical approaches to inoculate micropropagated sugar cane plants were Acetobacter diazotrophicus Effect of high sugar concentration on nitrogenase activity of Acetobacter diazotrophicus Physiology and dinitrogen fixation of Acetobacter diazotrophicus Plasmid contents and nif genes detection in Acetobacter diazotrophicus strains Analysis of nif and regulatory genes in Acetobacter diazotrophicus Characterization of genes involved in regulation of nitrogen fixation and ammonium sensing in Acetobacter diazotrophicus, an endophyte of sugarcane Molecular analysis of the chromosomal region encoding the nifA and nifB genes of Acetobacter diazotrophicus Characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus Identification of three genes encoding PII-like proteins in Gluconacetobacter diazotrophicus: studies of their role(s) in the control of nitrogen fixation Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus Genome-wide phylogenetic analysis of Gluconobacter, Acetobacter, and Gluconacetobacter: Genome-wide phylogenetic analysis of AAB 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) Draft genome sequence of Komagataeibacter rhaeticus strain AF1, a high producer of cellulose, isolated from Kombucha Tea Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem Acetic acid bacteria genomes reveal functional traits for adaptation to life in insect guts Draft genomic DNA sequence of the facultatively methylotrophic bacterium Acidomonas methanolica type strain MB58 Gluconacetobacter diazotrophicus Pal5 strain: selection and characterization of mutants deficient in nitrogen-fixation ability Protein expression profile of Gluconacetobacter diazotrophicus PAL5, a sugarcane endophytic plant growth-promoting bacterium A comparative proteomic analysis of Gluconacetobacter diazotrophicus PAL5 at exponential and stationary phases of cultures in the presence of high and low levels of inorganic nitrogen compound Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant Proteome of Gluconacetobacter diazotrophicus co-cultivated with sugarcane plantlets Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogenfixing endophyte Gluconacetobacter diazotrophicus Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5 Structural studies of an exopolysaccharide produced by Gluconacetobacter diazotrophicus Pal5 Gluconacetobacter diazotrophicus PAL5 possesses an active quorum sensing regulatory system Transcriptional regulation and signalpeptide-dependent secretion of exolevanase (LsdB) in the endophyte Gluconacetobacter diazotrophicus Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROSinduced nitrogenase inhibition in Gluconacetobacter diazotrophicus The bacterial superoxide dismutase and glutathione reductase are crucial for endophytic colonization of rice roots by Gluconacetobacter diazotrophicus PAL5 Influence of carbon and nitrogen sources on growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus Transfer RNA-dependent asparagine biosynthesis in Gluconacetobacter diazotrophicus and its influence on biological nitrogen fixation Nitrogen compounds in the apoplastic sap of sugarcane stem: some implications in the association with endophytes Inoculation of sugarcane with Pantoea sp. increases amino acid contents in shoot tissues; serine, alanine, glutamine and asparagine permit concomitantly ammonium excretion and nitrogenase activity of the bacterium Sustaining N2-dependent growth in the presence of CO NifA-and CooA-coordinated cowN expression sustains nitrogen fixation by Rhodobacter capsulatus in the presence of carbon monoxide Recent advances in nitrogen-fixing acetic acid bacteria Research progress and perspectives of nitrogen fixing bacterium, Gluconacetobacter diazotrophicus, in Monocot plants Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions Colonization of sorghum and wheat by seed inoculation with Gluconacetobacter diazotrophicus Monitoring the colonization of sugarcane and rice plants by the endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus marked with gfp and gusA reporter genes: Gfp marked G. diazotrophicus on rice Response of micropropagated sugarcane varieties to inoculation with endophytic diazotrophic bacteria Efeito da inoculaS cão de Acetobacter diazotrophicus em cana-de-aS c ucar (Saccharum spp) variedade SP70-1143, a partir de cultura de meristemas Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif-mutant strains Enhanced maize productivity by inoculation with diazotrophic bacteria Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media Ecological occurrence of Gluconacetobacter diazotrophicus and nitrogen-fixing Acetobacteraceae members: their possible role in plant growth promotion Evidence for a membranebound pyrroloquinoline quinone-linked glucose dehydrogenase in Acetobacter diazotrophicus Mineral phosphate solubilizing activity of Acetobacter diazotrophicus: a bacterium associated with sugar cane Solubilization of insoluble zinc compounds by Gluconacetobacter diazotrophicus and the detrimental action of zinc ion (Zn 2þ) and zinc chelates on root knot nematode Meloidogyne incognita Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc Assessing the zinc solubilization ability of Gluconacetobacter diazotrophicus in maize rhizosphere using labelled 65Zn compounds Mineral phosphate solubilization activity of Gluconacetobacter diazotrophicus under P-limitation and plant root environment Assessing the in vitro zinc solubilization potential and improving sugarcane growth by inoculating Gluconacetobacter diazotrophicus Zinc metal solubilization by Gluconacetobacter diazotrophicus and induction of pleomorphic cells Essential role of the czc determinant for cadmium, cobalt and zinc resistance in Gluconacetobacter diazotrophicus PAl 5 Gluconacetobacter diazotrophicus elicits a sugarcane defense response against a pathogenic bacteria Xanthomonas albilineans Antagonism of Gluconacetobacter diazotrophicus (a sugarcane endosymbiont) against Xanthomonas albilineans (pathogen) studied in alginate-immobilized sugarcane stalk tissues Antagonistic potential of N2-fixing Acetobacter diazotrophicus against Colletotrichum falcatum Went., a causal organism of red-rot of sugarcane Inoculation effects of Pseudomonas putida, Gluconacetobacter azotocaptans, and Azospirillum lipoferum on corn plant growth under greenhouse conditions In vitro suppression of soil borne pathogenic fungi and pyoluteorin production by Gluconacetobacter diazotrophicus Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans Signalling pathways mediating the association between sugarcane and endophytic diazotrophic bacteria: a genomic approach Members of the ethylene signalling pathway are regulated in sugarcane during the association with nitrogen-fixing endophytic bacteria Colonization behaviour of Gluconoacetobacter diazotrophicus in root-knot nematode (Meloidogyne incognita) infected and healthy cotton plants Validation list 149: list of new names and new combinations previously effectively, but not validly, published Asaia krungthepensis sp. nov., an acetic acid bacterium in the a-Proteobacteria Asaia lannaensis sp. nov., a new acetic acid bacterium in the alphaproteobacteria Acetic Acid Bacteria, newly emerging symbionts of insects Bacterial infections across the ants: frequency and prevalence of Wolbachia, Spiroplasma, and Asaia Field studies on response of sugar beet to microbial inoculants under graded nitrogen levels MEG A5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony methods Glucose metabolism and gluconic acid production by Acetobacter diazotrophicus Potential N 2 -fixation by sugarcane, Saccharum sp. in solution culture-I. Effect of NH 4 þ vs. NO 3 À , variety and nitrogen level Acetobacter diazotrophicus, an indoleacetic acid producing bacterium isolated from sugarcane cultivars of Mexico Influence of N fertilisation on the isolation of Acetobacter diazotrophicus and Herbaspirillum spp. from Indian sugarcane varieties Effect of inorganic N on the population, in vitro colonization and morphology of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus) Population dynamics of Gluconacetobacter diazotrophicus in sugarcane cultivars and its effect on plant growth Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp Nitrogen source effect on Gluconacetobacter diazotrophicus colonization of sugarcane (Saccharum spp Improving sugarcane growth and nutrient uptake by inoculating Gluconacetobacter diazotrophicus Nitrogen use efficiency of sugarcane in relation to its BNF potential and population of endophytic diazotrophs at different N levels Effects of diverse habitat biofertilizers on yield and nitrogen balance in plant-ratoon crop cycle of sugarcane in Subtropics Sprouting, survival and growth of young sugarcane (Saccharum officinarum L.) treated with diazotrophic bacteria (Gluconacetobacter diazotrophicus) The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants AvaliaS cão agronômica de variedades de cana-de-aS c ucar inoculadas com bact erias diazotr oficas e adubadas com nitrogênio Colonization of the nitrogen-fixing bacterium Gluconacetobacter diazotrophicus in a large number of Canadian corn plants Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane Functional production and secretion of the Gluconacetobacter diazotrophicus fructose-releasing exo-levanase (LsdB) in Pichia pastoris Antagonism among Gluconacetobacter diazotrophicus strains in culture media and in endophytic association Liquid formulations of Acetobacter diazotrophicus L1 and Herbaspirillum seropedicae J24 and their field trials on wheat Survival of endophytic bacteria in polymer-based inoculants and efficiency of their application to sugarcane Effect of diazotrophic bacterium inoculation and organic fertilization on yield of Champaka pineapple intercropped with irrigated sapota The authors wish to thank the Coordination of Improvement of Higher Education Personnel-CAPES, the National Council for Scientific and Technological Development-CNPq and the Carlos Chagas Foundation for Research Support of the State of Rio de Janeiro-FAPERJ for the scholarships. Finantial support and also scholarships from CNPq, project number 303125/2013-6 and also CNPq/INCT-FBN (Process No. 573828/2008-3).