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1.  Drought Tolerance Conferred to Sugarcane by Association with Gluconacetobacter diazotrophicus: A Transcriptomic View of Hormone Pathways 
PLoS ONE  2014;9(12):e114744.
Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant.
PMCID: PMC4260876  PMID: 25489849
2.  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
3.  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
4.  Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation 
Conifers predominantly occur on soils or in climates that are suboptimal for plant growth. This is generally attributed to symbioses with mycorrhizal fungi and to conifer adaptations, but recent experiments suggest that aboveground endophytic bacteria in conifers fix nitrogen (N) and affect host shoot tissue growth. Because most bacteria cannot be grown in the laboratory very little is known about conifer–endophyte associations in the wild. Pinus flexilis (limber pine) and Picea engelmannii (Engelmann spruce) growing in a subalpine, nutrient-limited environment are potential candidates for hosting endophytes with roles in N2 fixation and abiotic stress tolerance. We used 16S rRNA pyrosequencing to ask whether these conifers host a core of bacterial species that are consistently associated with conifer individuals and therefore potential mutualists. We found that while overall the endophyte communities clustered according to host species, both conifers were consistently dominated by the same phylotype, which made up 19–53% and 14–39% of the sequences in P. flexilis and P. engelmannii, respectively. This phylotype is related to Gluconacetobacter diazotrophicus and other N2 fixing acetic acid bacterial endophytes. The pattern observed for the P. flexilis and P. engelmannii needle microbiota—a small number of major species that are consistently associated with the host across individuals and species—is unprecedented for an endophyte community, and suggests a specialized beneficial endophyte function. One possibility is endophytic N fixation, which could help explain how conifers can grow in severely nitrogen-limited soil, and why some forest ecosystems accumulate more N than can be accounted for by known nitrogen input pathways.
PMCID: PMC4082182  PMID: 25071746
bacterial endophytes; 16S rRNA; conifers; Pinus; Picea; nitrogen; Acetobacteraceae; subalpine
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.  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.
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
7.  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
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.  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
10.  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
11.  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
12.  Genome Sequence of the Plant Growth Promoting Endophytic Bacterium Enterobacter sp. 638 
PLoS Genetics  2010;6(5):e1000943.
Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpa×deltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT–PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar as an energy feedstock on marginal, non-agricultural soils using endophytic bacteria as growth promoting agents.
Author Summary
Poplar is considered as the model tree species for the production of lignocellulosic biomass destined for biofuel production. The plant growth promoting endophytic bacterium Enterobacter sp. 638 can improve the growth of poplar on marginal soils by as much as 40%. This prompted us to sequence the genome of this strain and, via comparative genomics, identify functions essential for the successful colonization and endophytic association with its poplar host. Analysis of the genome sequence, combined with metabolite analysis and quantitative PCR, pointed to a remarkable interaction between Enterobacter sp. 638 and its poplar host with the endophyte responsible for the production of a phytohormone, and a precursor for another that poplar is unable to synthesize, and where the production of the plant growth promoting compounds depended on the presence of plant synthesized compounds, such as sucrose, in the growth medium. Our results provide the basis to better understanding the synergistic interactions between poplar and Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar on marginal, non-agricultural soils using endophytic bacteria such as Enterobacter sp. 638 as growth promoting agents.
PMCID: PMC2869309  PMID: 20485560
13.  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
14.  Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates▿  
Applied and Environmental Microbiology  2007;73(22):7259-7267.
Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.
PMCID: PMC2168197  PMID: 17905875
15.  Diversity of bacteria nesting the plant cover of north Sinai deserts, Egypt 
Journal of Advanced Research  2012;4(1):13-26.
North Sinai deserts were surveyed for the predominant plant cover and for the culturable bacteria nesting their roots and shoots. Among 43 plant species reported, 13 are perennial (e.g. Fagonia spp., Pancratium spp.) and 30 annuals (e.g. Bromus spp., Erodium spp.). Eleven species possessed rhizo-sheath, e.g. Cyperus capitatus, Panicum turgidum and Trisetaria koelerioides. Microbiological analyses demonstrated: the great diversity and richness of associated culturable bacteria, in particular nitrogen-fixing bacteria (diazotrophs); the majority of bacterial residents were of true and/or putative diazotrophic nature; the bacterial populations followed an increasing density gradient towards the root surfaces; sizeable populations were able to reside inside the root (endorhizosphere) and shoot (endophyllosphere) tissues. Three hundred bacterial isolates were secured from studied spheres. The majority of nitrogen-fixing bacilli isolates belonged to Bacillus megaterium,Bacillus pumilus, Bacillus polymexa,Bacillus macerans,Bacillus circulans and Bacillus licheniformis. The family Enterobacteriaceae represented by Enterobacter agglomerans,Enterobacter sackazakii, Enterobacter cloacae, Serratia adorifera,Serratia liquefaciens and Klebsiella oxytoca. The non-Enterobacteriaceae population was rich in Pantoae spp., Agrobacterium rdiobacter, Pseudomonas vesicularis, Pseudomonas putida, Stenotrophomonas maltophilia, Ochrobactrum anthropi, Sphingomonas paucimobilis and Chrysemonas luteola.Gluconacetobacter diazotrophicus were reported inside root and shoot tissues of a number of tested plants. The dense bacterial populations reported speak well to the very possible significant role played by the endophytic bacterial populations in the survival, in respect of nutrition and health, of existing plants. Such groups of diazotrophs are good candidates, as bio-preparates, to support the growth of future field crops grown in deserts of north Sinai and irrigated by the water of El-Salam canal.
PMCID: PMC4195458
North Sinai; Desert ecosystems; Xerophytes; Culturable bacteria; Rhizospheric microorganisms (RMOs); Diazotrophs; Rhizosheath
16.  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
17.  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
18.  Azoarcus sp. CIB, an Anaerobic Biodegrader of Aromatic Compounds Shows an Endophytic Lifestyle 
PLoS ONE  2014;9(10):e110771.
Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here.
Methodology/Principal Findings
Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe β-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches.
This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.
PMCID: PMC4207700  PMID: 25340341
19.  Plant Growth-Promoting Nitrogen-Fixing Enterobacteria Are in Association with Sugarcane Plants Growing in Guangxi, China 
Microbes and Environments  2012;27(4):391-398.
The current nitrogen fertilization for sugarcane production in Guangxi, the major sugarcane-producing area in China, is very high. We aim to reduce nitrogen fertilization and improve sugarcane production in Guangxi with the help of indigenous sugarcane-associated nitrogen-fixing bacteria. We initially obtained 196 fast-growing bacterial isolates associated with the main sugarcane cultivar ROC22 plants in fields using a nitrogen-deficient minimal medium and screened out 43 nitrogen-fixing isolates. Analysis of 16S rRNA gene sequences revealed that 42 of the 43 nitrogen-fixing isolates were affiliated with the genera Enterobacter and Klebsiella. Most of the nitrogen-fixing enterobacteria possessed two other plant growth-promoting activities of IAA production, siderophore production and phosphate solubilization. Two Enterobacter spp. strains of NN145S and NN143E isolated from rhizosphere soil and surface-sterilized roots, respectively, of the same ROC22 plant were used to inoculate micropropagated sugarcane plantlets. Both strains increased the biomass and nitrogen content of the sugarcane seedlings grown with nitrogen fertilization equivalent to 180 kg urea ha−1, the recommended nitrogen fertilization for ROC22 cane crops at the seedling stage. 15N isotope dilution assays demonstrated that biological nitrogen fixation contributed to plant growth promotion. These results suggested that indigenous nitrogen-fixing enterobacteria have the potential to fix N2 associated with sugarcane plants grown in fields in Guangxi and to improve sugarcane production.
PMCID: PMC4103546  PMID: 22510648
biological nitrogen fixation; enterobacteria; nifH; plant growth-promoting bacteria; sugarcane
20.  Genome sequencing of Sporisorium scitamineum provides insights into the pathogenic mechanisms of sugarcane smut 
BMC Genomics  2014;15(1):996.
Sugarcane smut can cause losses in cane yield and sugar content that range from 30% to total crop failure. Losses tend to increase with the passage of years. Sporisorium scitamineum is the fungus that causes sugarcane smut. This fungus has the potential to infect all sugarcane species unless a species is resistant to biotrophic fungal pathogens. However, it remains unclear how the fungus breaks through the cell walls of sugarcane and causes the formation of black or gray whip-like structures on the sugarcane plants.
Here, we report the first high-quality genome sequence of S. scitamineum assembled de novo with a contig N50 of 41 kb, a scaffold N50 of 884 kb and genome size 19.8 Mb, containing an estimated 6,636 genes. This phytopathogen can utilize a wide range of carbon and nitrogen sources. A reduced set of genes encoding plant cell wall hydrolytic enzymes leads to its biotrophic lifestyle, in which damage to the host should be minimized. As a bipolar mating fungus, a and b loci are linked and the mating-type locus segregates as a single locus. The S. scitamineum genome has only 6 G protein-coupled receptors (GPCRs) grouped into five classes, which are responsible for transducing extracellular signals into intracellular responses, however, the genome is without any PTH11-like GPCR. There are 192 virulence associated genes in the genome of S. scitamineum, among which 31 expressed in all the stages, which mainly encode for energy metabolism and redox of short-chain compound related enzymes. Sixty-eight candidates for secreted effector proteins (CSEPs) were found in the genome of S. scitamineum, and 32 of them expressed in the different stages of sugarcane infection, which are probably involved in infection and/or triggering defense responses. There are two non-ribosomal peptide synthetase (NRPS) gene clusters that are involved in the generation of ferrichrome and ferrichrome A, while the terpenes gene cluster is composed of three unknown function genes and seven biosynthesis related genes.
As a destructive pathogen to sugar industry, the S. scitamineum genome will facilitate future research on the genomic basis and the pathogenic mechanisms of sugarcane smut.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-996) contains supplementary material, which is available to authorized users.
PMCID: PMC4246466  PMID: 25406499
Sporisorium scitamineum, Sugarcane smut; Pathogenic mechanisms, G-protein coupled receptors, Carbohydrate degrading enzymes, Biotrophic properties, Candidates for secreted effector proteins, Secondary metabolic pathways
21.  Diazotrophic Burkholderia Species Associated with Field-Grown Maize and Sugarcane 
Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N2-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N2-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N2-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed.
PMCID: PMC1472400  PMID: 16672447
22.  Culturable bacterial endophytes isolated from Mangrove tree (Rhizophora apiculata Blume) enhance seedling growth in Rice 
Endophytic bacteria do have several potential applications in medicine and in other various sectors of biotechnology including agriculture. Bacterial endophytes need to be explored for their potential applications in agricultural biotechnology. One of the potential applications of bacterial endophytes in agricultural is to enhance the growth of the agricultural crops. Hence, this study was undertaken to explore the plant growth promoting potential application of bacterial endophytes.
The objective of this study was to examine the effect of endophytic bacteria from mangrove tree (Rhizophora apiculata Blume) for their efficacy in promoting seedling growth in rice.
Materials and Methods:
Eight endophytic bacterial isolates (EBIs) isolated from twig and petiole tissues of the mangrove were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequence homology. Separately, surface sterilized paddy seeds were treated with cell-free broth and cell suspension of the EBIs. Rice seedlings were analyzed by various bioassays and data was recorded.
The gene sequences of the isolates were closely related to two genera namely, Bacillus and Pantoea. Inoculation of EBIs from R. apiculata with rice seeds resulted in accelerated root and shoot growth with significant increase in chlorophyll content. Among the isolates, Pantoea ananatis (1MSE1) and Bacillus amyloliquefaciens (3MPE1) had shown predominance of activity. Endophytic invasion was recognized by the non-host by rapid accumulation of reactive oxygen species (ROS) and was counteracted by the production of hydrogen peroxide (H2O2) and lipid peroxide. The results demonstrated that EBIs from mangrove tree can increase the fitness of the rice seedlings under controlled conditions.
These research findings could be useful to enhance the seedling growth and could serve as foundation in further research on enhancing the growth of the rice crop using endophytic bacteria.
PMCID: PMC4121931  PMID: 25097431
16S ribosomal deoxyribonucleic acid; bacteria; bioprospecting; endophytes; mangrove; Rhizophora apiculata; rice
23.  Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1 
Applied and Environmental Microbiology  2012;78(21):7511-7518.
The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1::pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1::pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1::pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.
PMCID: PMC3485700  PMID: 22865062
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.  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

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