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1.  Indole-3-Acetic Acid Biosynthesis Is Deficient in Gluconacetobacter diazotrophicus Strains with Mutations in Cytochrome c Biogenesis Genes 
Journal of Bacteriology  2004;186(16):5384-5391.
Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only ∼6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA− phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.
PMCID: PMC490937  PMID: 15292139
2.  Gluconacetobacter diazotrophicus Elicits a Sugarcane Defense Response Against a Pathogenic Bacteria Xanthomonas albilineans 
Plant Signaling & Behavior  2006;1(5):265-273.
A new role for the plant growth-promoting nitrogen-fixing endophytic bacteria Gluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions. Living G.diazotrophicus possess and/or produce elicitor molecules which activate the sugarcane defense response resulting in the plant resistance to X. albilineans, in this particular case controlling the pathogen transmission to emerging agamic shoots. A total of 47 differentially expressed transcript derived fragments (TDFs) were identified by cDNA-AFLP. Transcripts showed significant homologies to genes of the ethylene signaling pathway (26%), proteins regulates by auxins (9%), β-1,3 Glucanase proteins (6%) and ubiquitin genes (4%), all major signaling mechanisms. Results point toward a form of induction of systemic resistance in sugarcane-G. diazotrophicus interactions which protect the plant against X. albilineans attack.
PMCID: PMC2634128  PMID: 19516988
Gluconacetobacter diazotrophicus; elicitors; sugarcane; Xanthomonas albilineans
3.  Two genome sequences of the same bacterial strain, Gluconacetobacter diazotrophicus PAl 5, suggest a new standard in genome sequence submission 
Standards in Genomic Sciences  2010;2(3):309-317.
Gluconacetobacter diazotrophicus PAl 5 is of agricultural significance due to its ability to provide fixed nitrogen to plants. Consequently, its genome sequence has been eagerly anticipated to enhance understanding of endophytic nitrogen fixation. Two groups have sequenced the PAl 5 genome from the same source (ATCC 49037), though the resulting sequences contain a surprisingly high number of differences. Therefore, an optical map of PAl 5 was constructed in order to determine which genome assembly more closely resembles the chromosomal DNA by aligning each sequence against a physical map of the genome. While one sequence aligned very well, over 98% of the second sequence contained numerous rearrangements. The many differences observed between these two genome sequences could be owing to either assembly errors or rapid evolutionary divergence. The extent of the differences derived from sequence assembly errors could be assessed if the raw sequencing reads were provided by both genome centers at the time of genome sequence submission. Hence, a new genome sequence standard is proposed whereby the investigator supplies the raw reads along with the closed sequence so that the community can make more accurate judgments on whether differences observed in a single stain may be of biological origin or are simply caused by differences in genome assembly procedures.
PMCID: PMC3035290  PMID: 21304715
Optical Mapping; Gluconacetobacter; chromosomal rearrangements
4.  Identification of Three Genes Encoding PII-Like Proteins in Gluconacetobacter diazotrophicus: Studies of Their Role(s) in the Control of Nitrogen Fixation 
Journal of Bacteriology  2003;185(19):5854-5861.
In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one PII protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other PII protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three PII protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three PII homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of PII proteins.
PMCID: PMC193954  PMID: 13129958
5.  Characterization of a Major Cluster of nif, fix, and Associated Genes in a Sugarcane Endophyte, Acetobacter diazotrophicus 
Journal of Bacteriology  2000;182(24):7088-7091.
A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus.
PMCID: PMC94840  PMID: 11092875
6.  Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROS-induced nitrogenase inhibition in Gluconacetobacter diazotrophicus 
Archives of Microbiology  2010;192(10):835-841.
Gluconacetobacter diazotrophicus, an endophyte isolated from sugarcane, is a strict aerobe that fixates N2. This process is catalyzed by nitrogenase and requires copious amounts of ATP. Nitrogenase activity is extremely sensitive to inhibition by oxygen and reactive oxygen species (ROS). However, the elevated oxidative metabolic rates required to sustain biological nitrogen fixation (BNF) may favor an increased production of ROS. Here, we explored this paradox and observed that ROS levels are, in fact, decreased in nitrogen-fixing cells due to the up-regulation of transcript levels of six ROS-detoxifying genes. A cluster analyses based on common expression patterns revealed the existence of a stable cluster with 99.8% similarity made up of the genes encoding the α-subunit of nitrogenase Mo–Fe protein (nifD), superoxide dismutase (sodA) and catalase type E (katE). Finally, nitrogenase activity was inhibited in a dose-dependent manner by paraquat, a redox cycler that increases cellular ROS levels. Our data revealed that ROS can strongly inhibit nitrogenase activity, and G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels resulting in a lower ROS generation. We suggest that careful controlled ROS production during this critical phase is an adaptive mechanism to allow nitrogen fixation.
PMCID: PMC2939349  PMID: 20697694
Gluconacetobacter diazotrophicus; Biological nitrogen fixation; Reactive oxygen species; Nitrogenase
7.  Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies 
Brazilian Journal of Microbiology  2009;40(4):866-878.
The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 105 cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.
PMCID: PMC3768561  PMID: 24031435
diazotrophic bacteria; ELISA; immunoquantification
8.  A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus 
Journal of Bacteriology  2004;186(15):5031-5039.
The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC to utilize plant sucrose. LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway. We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane. Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases. The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane. The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking. The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G. diazotrophicus structural gene. Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized. Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose. Replacement of Cys162 by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins. Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G. diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions. To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae.
PMCID: PMC451657  PMID: 15262940
9.  Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses 
Pedrosa, Fábio O. | Monteiro, Rose Adele | Wassem, Roseli | Cruz, Leonardo M. | Ayub, Ricardo A. | Colauto, Nelson B. | Fernandez, Maria Aparecida | Fungaro, Maria Helena P. | Grisard, Edmundo C. | Hungria, Mariangela | Madeira, Humberto M. F. | Nodari, Rubens O. | Osaku, Clarice A. | Petzl-Erler, Maria Luiza | Terenzi, Hernán | Vieira, Luiz G. E. | Steffens, Maria Berenice R. | Weiss, Vinicius A. | Pereira, Luiz F. P. | Almeida, Marina I. M. | Alves, Lysangela R. | Marin, Anelis | Araujo, Luiza Maria | Balsanelli, Eduardo | Baura, Valter A. | Chubatsu, Leda S. | Faoro, Helisson | Favetti, Augusto | Friedermann, Geraldo | Glienke, Chirlei | Karp, Susan | Kava-Cordeiro, Vanessa | Raittz, Roberto T. | Ramos, Humberto J. O. | Ribeiro, Enilze Maria S. F. | Rigo, Liu Un | Rocha, Saul N. | Schwab, Stefan | Silva, Anilda G. | Souza, Eliel M. | Tadra-Sfeir, Michelle Z. | Torres, Rodrigo A. | Dabul, Audrei N. G. | Soares, Maria Albertina M. | Gasques, Luciano S. | Gimenes, Ciela C. T. | Valle, Juliana S. | Ciferri, Ricardo R. | Correa, Luiz C. | Murace, Norma K. | Pamphile, João A. | Patussi, Eliana Valéria | Prioli, Alberto J. | Prioli, Sonia Maria A. | Rocha, Carmem Lúcia M. S. C. | Arantes, Olívia Márcia N. | Furlaneto, Márcia Cristina | Godoy, Leandro P. | Oliveira, Carlos E. C. | Satori, Daniele | Vilas-Boas, Laurival A. | Watanabe, Maria Angélica E. | Dambros, Bibiana Paula | Guerra, Miguel P. | Mathioni, Sandra Marisa | Santos, Karine Louise | Steindel, Mario | Vernal, Javier | Barcellos, Fernando G. | Campo, Rubens J. | Chueire, Ligia Maria O. | Nicolás, Marisa Fabiana | Pereira-Ferrari, Lilian | da Conceição Silva, José L. | Gioppo, Nereida M. R. | Margarido, Vladimir P. | Menck-Soares, Maria Amélia | Pinto, Fabiana Gisele S. | Simão, Rita de Cássia G. | Takahashi, Elizabete K. | Yates, Marshall G. | Souza, Emanuel M. | Richardson, Paul M.
PLoS Genetics  2011;7(5):e1002064.
The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.
Author Summary
In this work we describe the genome of H. seropedicae SmR1, a bacterium capable of fixing nitrogen and promoting the growth of important plant crops such as maize, rice, and sugar cane. Several investigations have shown that H. seropedicae supplies fixed nitrogen to the associated plant and increases grain productivity up to 50%. In the genome of H. seropedicae, we identified all the genes involved in the nitrogen fixation process and its regulation and, in addition, genes potentially involved in the establishment of efficient interaction with the host plant. Our analyses also revealed that this bacterium has a highly versatile metabolism capable of synthesizing and degrading a large number of organic and inorganic compounds. We believe that the knowledge of the genome of this bacterium will direct research to a better understanding of this important endophytic organism and allow the construction of new strains with enhanced agronomic efficiency.
PMCID: PMC3093359  PMID: 21589895
10.  Response of the Endophytic Diazotroph Gluconacetobacter diazotrophicus on Solid Media to Changes in Atmospheric Partial O2 Pressure 
Applied and Environmental Microbiology  2001;67(10):4694-4700.
Gluconacetobacter diazotrophicus is an N2-fixing endophyte isolated from sugarcane. G. diazotrophicus was grown on solid medium at atmospheric partial O2 pressures (pO2) of 10, 20, and 30 kPa for 5 to 6 days. Using a flowthrough gas exchange system, nitrogenase activity and respiration rate were then measured at a range of atmospheric pO2 (5 to 60 kPa). Nitrogenase activity was measured by H2 evolution in N2-O2 and in Ar-O2, and respiration rate was measured by CO2 evolution in N2-O2. To validate the use of H2 production as an assay for nitrogenase activity, a non-N2-fixing (Nif−) mutant of G. diazotrophicus was tested and found to have a low rate of uptake hydrogenase (Hup+) activity (0.016 ±  0.009 μmol of H2 1010 cells−1 h−1) when incubated in an atmosphere enriched in H2. However, Hup+ activity was not detectable under the normal assay conditions used in our experiments. G. diazotrophicus fixed nitrogen at all atmospheric pO2 tested. However, when the assay atmospheric pO2 was below the level at which the colonies had been grown, nitrogenase activity was decreased. Optimal atmospheric pO2 for nitrogenase activity was 0 to 20 kPa above the pO2 at which the bacteria had been grown. As atmospheric pO2 was increased in 10-kPa steps to the highest levels (40 to 60 kPa), nitrogenase activity decreased in a stepwise manner. Despite the decrease in nitrogenase activity as atmospheric pO2 was increased, respiration rate increased marginally. A large single-step increase in atmospheric pO2 from 20 to 60 kPa caused a rapid 84% decrease in nitrogenase activity. However, upon returning to 20 kPa of O2, 80% of nitrogenase activity was recovered within 10 min, indicating a “switch-off/switch-on” O2 protection mechanism of nitrogenase activity. Our study demonstrates that colonies of G. diazotrophicus can fix N2 at a wide range of atmospheric pO2 and can adapt to maintain nitrogenase activity in response to both long-term and short-term changes in atmospheric pO2.
PMCID: PMC93221  PMID: 11571174
11.  The genome of the endophytic bacterium H. frisingense GSF30T identifies diverse strategies in the Herbaspirillum genus to interact with plants 
The diazotrophic, bacterial endophyte Herbaspirillum frisingense GSF30T has been identified in biomass grasses grown in temperate climate, including the highly nitrogen-efficient grass Miscanthus. Its genome was annotated and compared with related Herbaspirillum species from diverse habitats, including H. seropedicae, and further well-characterized endophytes. The analysis revealed that Herbaspirillum frisingense lacks a type III secretion system that is present in some related Herbaspirillum grass endophytes. Together with the lack of components of the type II secretion system, the genomic inventory indicates distinct interaction scenarios of endophytic Herbaspirillum strains with plants. Differences in respiration, carbon, nitrogen and cell wall metabolism among Herbaspirillum isolates partially correlate with their different habitats. Herbaspirillum frisingense is closely related to strains isolated from the rhizosphere of phragmites and from well water, but these lack nitrogen fixation and metabolism genes. Within grass endophytes, the high diversity in their genomic inventory suggests that even individual plant species provide distinct, highly diverse metabolic niches for successful endophyte-plant associations.
PMCID: PMC3695564  PMID: 23825472
microbe; diazotroph; nitrogen fixation; plant associated bacteria; plant growth promoting bacteria
12.  Complete Genome Sequence of Enterobacter sp. Strain R4-368, an Endophytic N-Fixing Gammaproteobacterium Isolated from Surface-Sterilized Roots of Jatropha curcas L. 
Genome Announcements  2013;1(4):e00544-13.
Enterobacter sp. strain R4-368 is one of the few characterized Jatropha endophytic diazotrophic bacteria and was isolated from surface-sterilized roots. This bacterium shows strong growth-promoting effects, being able to increase plant biomass and seed yields. Enterobacter sp. R4-368 is the second fully sequenced diazotrophic Enterobacter species. The sequence information shall facilitate the elucidation of the molecular mechanisms of plant growth promotion, nitrogen fixation in nonlegume plant species, and evolution of biological nitrogen fixation systems.
PMCID: PMC3731841  PMID: 23908287
13.  Genome Sequence of Enterobacter sp. Strain SP1, an Endophytic Nitrogen-Fixing Bacterium Isolated from Sugarcane 
Journal of Bacteriology  2012;194(24):6963-6964.
Enterobacter sp. strain SP1 is an endophytic nitrogen-fixing bacterium isolated from a sugarcane stem and can promote plant growth. The draft genome sequence of strain SP1 presented here will promote comparative genomic studies to determine the genetic background of interactions between endophytic enterobacteria and plants.
PMCID: PMC3510633  PMID: 23209221
14.  Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria 
BMC Genomics  2010;11(Suppl 5):S7.
G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms.
Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved “fer2” domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface.
The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.
PMCID: PMC3045800  PMID: 21210973
15.  Energy Generation by Extracellular Aldose Oxidation in N2-Fixing Gluconacetobacter diazotrophicus 
Gluconacetobacter diazotrophicus PAL3 was grown in a chemostat with N2 and mixtures of xylose and gluconate. Xylose was oxidized to xylonate, which was accumulated in the culture supernatants. Biomass yields and carbon from gluconate incorporated into biomass increased with the rate of xylose oxidation. By using metabolic balances it is demonstrated that extracellular xylose oxidation led N2-fixing G. diazotrophicus cultures to increase the efficiency of energy generation.
PMCID: PMC123872  PMID: 11916732
16.  Polyphasic characterization of Gluconacetobacter diazotrophicus isolates obtained from different sugarcane varieties 
Brazilian Journal of Microbiology  2008;39(4):718-723.
A polyphasic approach was applied to characterize 35 G. diazotrophicus isolates obtained from sugarcane varieties cultivated in Brazil. The isolates were analyzed by phenotypic (use of different carbon sources) and genotypic tests (ARDRA and RISA–RFLP techniques). Variability among the isolates was observed in relation to the carbon source use preference. Glucose and sucrose were used by all isolates in contrast to myo-inositol, galactose and ribose that were not metabolized. The results of the analysis showed the presence of two groups clustered at 68% of similarity. The genetic distance was higher when RISA-RFLP analysis was used. Analysis of 16S rDNA sequences from isolates showed that all of them belonged to the G. diazotrophicus species. Neither effect of the plant part nor sugarcane variety was observed during the cluster analysis. The observed metabolic and genetic variability will be helpful during the strain selection studies for sugarcane inoculation in association with sugarcane breeding programs.
PMCID: PMC3768463  PMID: 24031296
biological nitrogen fixation; genetic diversity; plant-bacteria interaction
17.  Further Evidence that the N(inf2)-Fixing Endophytic Bacterium from the Intercellular Spaces of Sugarcane Stems Is Acetobacter diazotrophicus 
Nitrogen-fixing bacteria, isolated from the sugar solution in intercellular spaces of sugarcane stems, were compared with the type strain of Acetobacter diazotrophicus (PAL-5) and found to be congruent with it in all characters studied. These characters were 37 morphological and biochemical tests, cellular fatty acid composition, and nitrogenase activity. The nitrogenase activity was measured by acetylene reduction and H(inf2) evolution and found to be unusual in that the H(inf2) evolution was suppressed much less than expected by high concentrations of acetylene.
PMCID: PMC1388444  PMID: 16535026
18.  Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus, a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b, and Cytochrome c▿  
Journal of Bacteriology  2010;192(21):5718-5724.
Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.
PMCID: PMC2953696  PMID: 20802042
19.  Coffea arabica L., a new host plant for Acetobacter diazotrophicus, and isolation of other nitrogen-fixing acetobacteria. 
Acetobacter diazotrophicus was isolated from coffee plant tissues and from rhizosphere soils. Isolation frequencies ranged from 15 to 40% and were dependent on soil pH. Attempts to isolate this bacterial species from coffee fruit, from inside vesicular-arbuscular mycorrhizal fungi spores, or from mealybugs (Planococcus citri) associated with coffee plants were not successful. Other acid-producing diazotrophic bacteria were recovered with frequencies of 20% from the coffee rhizosphere. These N2-fixing isolates had some features in common with the genus Acetobacter but should not be assigned to the species Acetobacter diazotrophicus because they differed from A. diazotrophicus in morphological and biochemical traits and were largely divergent in electrophoretic mobility patterns of metabolic enzymes at coefficients of genetic distance as high as 0.950. In addition, these N2-fixing acetobacteria differed in the small-subunit rRNA restriction fragment length polymorphism patterns obtained with EcoRI, and they exhibited very low DNA-DNA homology levels, ranging from 11 to 15% with the A. diazotrophicus reference strain PAI 5T. Thus, some of the diazotrophic acetobacteria recovered from the rhizosphere of coffee plants may be regarded as N2-fixing species of the genus Acetobacter other than A. diazotrophicus. Endophytic diazotrophic bacteria may be more prevalent than previously thought, and perhaps there are many more potentially beneficial N2-fixing bacteria which can be isolated from other agronomically important crops.
PMCID: PMC168673  PMID: 9293018
20.  Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice 
PLoS Genetics  2008;4(7):e1000141.
We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.
Author Summary
Bacterial endophytes are capable of inhabiting the living tissues of plants without causing them significant harm. Klebsiella pneumoniae 342 (Kp342) is a model for this plant host-bacterial association, in part due to its capacity to colonize in high numbers the interior of plants including wheat and maize, two of the most important crops in the world. Kp342 possesses the ability to capture atmospheric nitrogen gas and turn it into an organic form (a process known as nitrogen fixation), of which part may be used as fertilizer by its plant host. Here, we describe the genome sequence and analysis of this model endophyte. When the Kp342 genome is compared to the genome of a closely related pathogenic relative, we can begin to surmise that its preference to engage in a harmonious relationship with plants is a result of many interacting factors. These include differences in its protein secretion systems, the manner in which its genes are regulated, and its ability to sense and respond to its environment. The study of endophytes is increasing in intensity due to the roles they may play in multiple biotechnological applications, including enhancing crop growth and nutrition, bioremediation, and development of plant-derived products and biofuels.
PMCID: PMC2453333  PMID: 18654632
21.  Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510 
We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N2 fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C4-dicarboxylate during its symbiotic relationship with the host plant.
PMCID: PMC2818188  PMID: 20047946
Azospirillum; endophyte; rice plant; N2 fixation; plant hormone
22.  Genetic Structure of Acetobacter diazotrophicus Populations and Identification of a New Genetically Distant Group 
A total of 55 isolates of Acetobacter diazotrophicus recovered from diverse sucrose-rich host plants and from mealybugs associated with sugarcane plants were characterized by the electrophoretic mobilities of 12 metabolic enzymes. We identified seven different electrophoretic types (ETs), six of which are closely related within a genetic distance of 0.195 and exhibit high DNA-DNA homology. The seventh ET was largely divergent, separated at a genetic distance of 0.53, and had only 54% DNA homology to the reference strain. Strains corresponding to ET 7 could represent a distinct nitrogen-fixing species of the genus Acetobacter. More genetic diversity was found in isolates from Brazil than in those from Mexico, probably due to the very different crop nitrogen fertilization levels used.
PMCID: PMC1388556  PMID: 16535102
23.  Metabolic potential of endophytic bacteria☆ 
Current Opinion in Biotechnology  2014;27(100):30-37.
Graphical abstract
•Endophytes are a source of a plethora of biologically active substances.•Endophyte-associated metabolites may be needed for the interaction with the plant.•Some metabolites are produced jointly by plant and endophytes.•Endophytes may stimulate or alter metabolite production by the plant.•Metabolite functions include signalling and communication, nutrient acquisition and defense.
The bacterial endophytic microbiome promotes plant growth and health and beneficial effects are in many cases mediated and characterized by metabolic interactions. Recent advances have been made in regard to metabolite production by plant microsymbionts showing that they may produce a range of different types of metabolites. These substances play a role in defense and competition, but may also be needed for specific interaction and communication with the plant host. Furthermore, few examples of bilateral metabolite production are known and endophytes may modulate plant metabolite synthesis as well. We have just started to understand such metabolic interactions between plants and endophytes, however, further research is needed to more efficiently make use of beneficial plant-microbe interactions and to reduce pathogen infestation as well as to reveal novel bioactive substances of commercial interest.
PMCID: PMC4045207  PMID: 24863894
24.  Epicoccum nigrum P16, a Sugarcane Endophyte, Produces Antifungal Compounds and Induces Root Growth 
PLoS ONE  2012;7(6):e36826.
Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology.
Methodology/Principal Findings
We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment.
Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.
PMCID: PMC3366970  PMID: 22675473
25.  Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes 
BMC Genomics  2012;13:162.
The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes.
Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions.
Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.
PMCID: PMC3464626  PMID: 22554235

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