A comparative proteomic analysis of Gluconacetobacter diazotrophicus PAL5 at exponential and stationary phases of cultures in the presence of high and low levels of inorganic nitrogen compound

A proteomic view of G. diazotrophicus PAL5 at the exponential (E) and stationary phases (S) of cultures in the presence of low (L) and high levels (H) of combined nitrogen is presented. The proteomes analyzed on 2D-gels showed 131 proteins (42E+32S+29H+28L) differentially expressed by G. diazotrophicus, from which 46 were identified by combining mass spectrometry and bioinformatics tools. Proteins related to cofactor, energy and DNA metabolisms and cytoplasmic pH homeostasis were differentially expressed in E growth phase, under L and H conditions, in line with the high metabolic rate of the cells and the low pH of the media. Proteins most abundant in S-phase cells were stress associated and transporters plus transferases in agreement with the general phenomenon that binding protein-dependent systems are induced under nutrient limitation as part of hunger response. Cells grown in L condition produced nitrogen-fixation accessory proteins with roles in biosynthesis and stabilization of the nitrogenase complex plus proteins for protection of the nitrogenases from O(2)-induced inactivation. Proteins of the cell wall biogenesis apparatus were also expressed under nitrogen limitation and might function in the reshaping of the nitrogen-fixing G. diazotrophicus cells previously described. Genes whose protein products were detected in our analysis were mapped onto the chromosome and, based on the tendency of functionally related bacterial genes to cluster, we identified genes of particular pathways that could be organized in operons and are co-regulated. These results showed the great potential of proteomics to describe events in G. diazotrophicus cells by looking at proteins expressed under distinct growth conditions.     

Quantitative proteomic analysis of the interaction between the endophytic plant-growth-promoting bacterium Gluconacetobacter diazotrophicus and sugarcane

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium that colonizes sugarcane. In order to investigate molecular aspects of the G. diazotrophicus-sugarcane interaction, we performed a quantitative mass spectrometry-based proteomic analysis by (15)N metabolic labeling of bacteria, root samples, and co-cultures. Overall, more than 400 proteins were analyzed and 78 were differentially expressed between the plant-bacterium interaction model and control cultures. A comparative analysis of the G. diazotrophicus in interaction with two distinct genotypes of sugarcane, SP70-1143 and Chunee, revealed proteins with fundamental roles in cellular recognition. G. diazotrophicus presented proteins involved in adaptation to atypical conditions and signaling systems during the interaction with both genotypes. However, SP70-1143 and Chunee, sugarcane genotypes with high and low contribution of biological nitrogen fixation, showed divergent responses in contact with G. diazotrophicus. The SP70-1143 genotype overexpressed proteins from signaling cascades and one from a lipid metabolism pathway, whereas Chunee differentially synthesized proteins involved in chromatin remodeling and protein degradation pathways. In addition, we have identified 30 bacterial proteins in the roots of the plant samples; from those, nine were specifically induced by plant signals. This is the first quantitative proteomic analysis of a bacterium-plant interaction, which generated insights into early signaling of the G. diazotrophicus-sugarcane interaction.     

Solubilization of insoluble zinc compounds by Gluconacetobacter diazotrophicus and the detrimental action of zinc ion (Zn2+) and zinc chelates on root knot nematode Meloidogyne incognita

AIM: To examine the zinc (Zn) solubilization potential and nematicidal properties of Gluconacetobacter diazotrophicus. METHODS AND RESults: Atomic Absorption Spectrophotometer, Differential Pulse Polarography and Gas Chromatography Coupled Mass Spectrometry were used to estimate the total Zn and Zn(2+) ions and identify the organic acids present in the culture supernatants. The effect of culture filtrate of Zn-amended G. diazotrophicus PAl5 on Meloidogyne incognita in tomato was examined under gnotobiotic conditions. Gluconacetobacter diazotrophicus PAl5 effectively solubilized the Zn compounds tested and 5-ketogluconic acid was identified as the major organic acid aiding the solubilization of zinc oxide. The presence of Zn compounds in the culture filtrates of G. diazotrophicus enhanced the mortality and reduced the root penetration of M. incognita under in vitro conditions. CONCLUSIONS: 5-ketogluconic acid produced by G. diazotrophicus mediated the solubilization process and the available Zn(2+) ions enhanced the nematicidal activity of G. diazotrophicus against M. incognita. SIGNIFICANCE AND IMPACT OF THE STUDY: Zn solubilization and enhanced nematicidal activity of Zn-amended G. diazotrophicus provides the possibility of exploiting it as a plant growth promoting bacteria.     

Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate

Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

Proteomic analysis of differential protein expression in platelets of septic patients

Sepsis is one of the major health problems all over the world. Early diagnostic of sepsis is an attractive strategy to decrease the mortality of septic patients. However, an effective biomarker that fulfills all the necessary requirements for the accurate characterization of sepsis is still unavailable until now. In this study, the 2-DE technique followed by mass spectrometry and a database search was used for searching and identifying the differential expressed proteins in platelets between septic patients and paired healthy controls. Platelet 2-DE profiles of septic patients and paired healthy controls with high resolution and reproducibility were obtained. Differential platelet 2-DE profiles between septic patients and paired healthy controls were established. Differential protein spots between normal healthy volunteers and septic patients from platelet 2-DE profiles were identified by 2-DE followed with mass spectrometry and a database search. Five proteins with increased expression were identified between septic patients and healthy controls from platelet samples. These up-expressed proteins were EF-hand calcium-binding domain-containing protein 7, actin, interleukin-1β, glycoprotein IX, and glycoprotein IIB. Sepsis induces a complex regulation of platelet protein changes. Our study highlights the important role of these differential expressed proteins in sepsis, which deserve further research as potential candidates for therapeutic strategies. Furthermore, our research is beneficial for the future developments of sepsis diagnosis and therapy.     

Isolation and characterizations of oxalate-binding proteins in the kidney

Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) to resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed "L-x(3,5)-R-x(2)-[AGILPV]" as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone modulators.     

Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two-dimensional electrophoresis

Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.     

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene codes for 130-kDa toxin protein

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.     

Energy generation by extracellular aldose oxidation in N(2)-fixing Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus PAL3 was grown in a chemostat with N(2) 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 N(2)-fixing G. diazotrophicus cultures to increase the efficiency of energy generation.     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems,and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intraceliularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers,we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus,with minimal or zero inputs.     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.     

Proteins identified as targets of the acyl glucuronide metabolite of mycophenolic acid in kidney tissue from mycophenolate mofetil treated rats

Covalent binding of the acyl glucuronide (AcMPAG) metabolite of the immunosuppressant mycophenolic acid (MPA) to proteins is considered a possible initiating event for organ toxicity. Since the kidney is involved in the formation and excretion of AcMPAG, it can be hypothesized that this tissue may be exposed to relatively high concentrations of this metabolite and would, therefore, be a particularly suitable organ to investigate AcMPAG protein targets. In the present study we identified potential AcMPAG target proteins in kidney tissues from Wistar rats treated with mycophenolate mofetil (40 mg/kg/day over 21 days). Proteins were separated by 2-DE and covalent protein adducts were detected by Western blotting with an antibody specific for MPA/AcMPAG. The corresponding coomassie blue stained proteins from parallel gels were subjected to in-gel tryptic digestion and peptides were characterized on a Q-TOF Ultima Global. The protein targets were further verified by immunoprecipitation with anti-MPA/AcMPAG antibody to purify the modified proteins followed by 1-DE and MS analysis. Database searches revealed several AcMPAG target proteins that could be related to ultrastructural abnormalities, metabolic effects, and altered oxidative stress/detoxification responses. Predominately cytosolic proteins such as selenium binding protein, protein disulfide isomerase, aldehyde dehydrogenase, triosephosphate isomerase, and kidney aminoacylase were involved in adduct formation. Two cytoskeletal proteins tropomyosin 1 and 4 as well as the antioxidant proteins peroxiredoxin 3 and 6 were also targets of AcMPAG. Functional consequences from these protein modifications remain to be demonstrated.     

Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes

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 approximately 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.     

Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes

Salinity is one of the major environmental constraints limiting yield of crop plants in many semi-arid and arid regions around the world. To understand responses in soybean seedling to salt stress at proteomic level, the extracted proteins from seedling leaves of salt-sensitive genotype Jackson and salt-tolerant genotype Lee 68 under 150 mM NaCl stress for 1, 12, 72 and 144 h, respectively, were analyzed by 2-DE. Approximately 800 protein spots were detected on 2-DE gels. Among them, 91 were found to be differently expressed, with 78 being successfully identified by MALDI-TOF-TOF. The identified proteins were involved in 14 metabolic pathways and cellular processes. Based on most of the 78 salt-responsive proteins, a salt stress-responsive protein network was proposed. This network consisted of several functional components, including balancing between ROS production and scavenging, accelerated proteolysis and reduced biosynthesis of proteins, impaired photosynthesis, abundant energy supply and enhanced biosynthesis of ethylene. Salt-tolerant genotype Lee 68 possessed the ability of higher ROS scavenging, more abundant energy supply and ethylene production, and stronger photosynthesis than salt-sensitive genotype Jackson under salt stress, which may be the major reasons why it is more salt-tolerant than Jackson.     

Web-accessible proteome databases for microbial research

The analysis of proteomes of biological organisms represents a major challenge of the post-genome era. Classical proteomics combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) for the identification of proteins. Novel technologies such as isotope coded affinity tag (ICAT)-liquid chromatography/mass spectrometry (LC/MS) open new insights into protein alterations. The vast amount and diverse types of proteomic data require adequate web-accessible computational and database technologies for storage, integration, dissemination, analysis and visualization. A proteome database system (http://www.mpiib-berlin.mpg.de/2D-PAGE) for microbial research has been constructed which integrates 2-DE/MS, ICAT-LC/MS and functional classification data of proteins with genomic, metabolic and other biological knowledge sources. The two-dimensional polyacrylamide gel electrophoresis database delivers experimental data on microbial proteins including mass spectra for the validation of protein identification. The ICAT-LC/MS database comprises experimental data for protein alterations of mycobacterial strains BCG vs. H37Rv. By formulating complex queries within a functional protein classification database "FUNC_CLASS" for Mycobacterium tuberculosis and Helicobacter pylori the researcher can gather precise information on genes, proteins, protein classes and metabolic pathways. The use of the R language in the database architecture allows high-level data analysis and visualization to be performed "on-the-fly". The database system is centrally administrated, and investigators without specific bioinformatic competence in database construction can submit their data. The database system also serves as a template for a prototype of a European Proteome Database of Pathogenic Bacteria. Currently, the database system includes proteome information for six strains of microorganisms.     

Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions.

Sugar cane (Saccharum spp.) variety SP 70-1143 was inoculated with Gluconacetobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments. In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted. After 10 d there was extensive anatomical evidence of endophytic colonization by G. diazotrophicus, particularly in lower stems, where high numbers of bacteria were visible within some of the xylem vessels. The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G. diazotrophicus. On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these 'wounds' bacteria were seen colonizing the xylem and intercellular spaces. Bacteria were also occasionally seen entering leaves via damaged stomata, and subsequently colonizing sub-stomatal cavities and intercellular spaces. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion. In experiment 2, stems of 5-week-old greenhouse-grown plants were inoculated by injection with a suspension of G. diazotrophicus containing 10(8) bacteria ml(-1). No hypersensitive response (HR) was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment (7 d). Close to the point of inoculation, G. diazotrophicus cells were observed within the protoxylem and the xylem parenchyma, where they were surrounded by fibrillar material that stained light-green with toluidine blue. In leaf samples taken up to 4 cm from the inoculation points, G. diazotrophicus cells were mainly found within the metaxylem, where they were surrounded by a light green-staining material. The bacteria were growing in relatively low numbers adjacent to the xylem cell walls, and they were separated from the host-derived material by electron-transparent 'haloes' that contained material that reacted with the G. diazotrophicus antibody.     

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

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