RESUMO
The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Gluconacetobacter/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Periplásmicas/metabolismo , Serina Endopeptidases/metabolismo , Cloreto de Sódio/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Gluconacetobacter/enzimologia , Gluconacetobacter/genética , Proteínas de Choque Térmico/genética , Ferro/metabolismo , Peptídeo Hidrolases/genética , Proteínas Periplásmicas/genética , Serina Endopeptidases/genéticaRESUMO
Pecan nutshell is an abundant waste with a high content of carbohydrates. According to its chemical composition, pecan nutshell could be used as carbon source for Gluconacetobacter entanii, a bacterium that produces cellulose with high purity and nanometric characteristics. Bacterial cellulose (BC) was obtained from a static culture medium using pecan nutshell as carbon source and saccharose as control. Results showed that the pecan nutshell could be used as carbon source for production of BC. The cellulose yield ranged around 2.816 ± 0.040 g/L for 28 days. The morphological, structural and chemical properties of the cellulose produced were similar to those reported for others BC. The spectroscopic characterization indicated the chemical functionalization of BC and the reduction of its crystallinity. The production of BC with G. entanii using pecan nutshell as carbon source, is the first report. The BC could have potential use in chemical functionalization and in the preparation of biocomposites.
Assuntos
Carya/química , Celulose/biossíntese , Celulose/química , Gluconacetobacter/metabolismo , Nozes/química , Celulose/isolamento & purificaçãoRESUMO
AIMS: Obtain varieties of Gluconacetobacter hansenii from original strain ATCC 23729 with greater efficiency to produce bacterial cellulose (BC) membrane with better dry mass yield for application as support of sustained antimicrobials' drug release. METHODS AND RESULTS: Application of different chemical and physical conditions (pH, temperature and UV light exposure) to obtain different G. hansenii varieties with high capacity to produce BC membranes. Characterization of the G. hansenii variants was performed by scanning electron microscopy (SEM) and optical microscopy of the colony-forming units. BC membrane produced was characterized by SEM, infrared spectroscopy and X-ray diffraction. The BC produced by variants isolated after incubation at 35°C showed elevated dry mass yield and high capacity of retention and sustained release of ceftriaxone antibiotic with the produced BC by original G. hansenii ATCC 23769 strain subjected to incubation at 28°C and with commercial BC. CONCLUSION: The application of different chemical and physical conditions constitutes an important method to obtain varieties of micro-organisms with dissimilar metabolism advantageous in relation to the original strain in the BC production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the importance of in vivo studies for the application, in medicine, of BC membranes as support for antimicrobial-sustained release for the skin wound treatment.
Assuntos
Anti-Infecciosos/farmacocinética , Celulose , Preparações de Ação Retardada/química , Gluconacetobacter , Ceftriaxona/farmacocinética , Celulose/química , Celulose/metabolismo , Celulose/ultraestrutura , Gluconacetobacter/química , Gluconacetobacter/metabolismo , Microscopia Eletrônica de Varredura , Difração de Raios XRESUMO
To reduce the cost of obtaining bacterial cellulose, acidic by-products of the alcohol and dairy industries were used without any pretreatment or addition of other nitrogen sources. Studies have shown that the greatest accumulation of bacterial cellulose (6.19g/L) occurs on wheat thin stillage for 3 days of cultivation under dynamic conditions, which is almost 3 times higher than on standard Hestrin and Schramm medium (2.14g/L). The use of whey as a nutrient medium makes it possible to obtain 5.45g/L bacterial cellulose under similar conditions of cultivation. It is established that the pH of the medium during the growth of Gluconacetobacter sucrofermentans B-11267 depends on the feedstock used and its initial value. By culturing the bacterium on thin stillage and whey, there is a decrease in the acidity of the waste. It is shown that the infrared spectra of bacterial cellulose obtained in a variety of environments have a similar character, but we found differences in the micromorphology and crystallinity of the resulting biopolymer.
Assuntos
Celulose/biossíntese , Gluconacetobacter/metabolismo , Microbiologia Industrial/métodos , Resíduos/análise , Meios de Cultura/economia , Meios de Cultura/metabolismo , Etanol/metabolismo , Indústria Alimentícia , Gluconacetobacter/crescimento & desenvolvimento , Microbiologia Industrial/economia , Triticum/metabolismo , Triticum/microbiologia , Resíduos/economiaRESUMO
Abstract To reduce the cost of obtaining bacterial cellulose, acidic by-products of the alcohol and dairy industries were used without any pretreatment or addition of other nitrogen sources. Studies have shown that the greatest accumulation of bacterial cellulose (6.19 g/L) occurs on wheat thin stillage for 3 days of cultivation under dynamic conditions, which is almost 3 times higher than on standard Hestrin and Schramm medium (2.14 g/L). The use of whey as a nutrient medium makes it possible to obtain 5.45 g/L bacterial cellulose under similar conditions of cultivation. It is established that the pH of the medium during the growth of Gluconacetobacter sucrofermentans B-11267 depends on the feedstock used and its initial value. By culturing the bacterium on thin stillage and whey, there is a decrease in the acidity of the waste. It is shown that the infrared spectra of bacterial cellulose obtained in a variety of environments have a similar character, but we found differences in the micromorphology and crystallinity of the resulting biopolymer.
Assuntos
Resíduos/análise , Microbiologia Industrial/métodos , Celulose/biossíntese , Gluconacetobacter/metabolismo , Resíduos/economia , Triticum/metabolismo , Triticum/microbiologia , Microbiologia Industrial/economia , Indústria Alimentícia , Meios de Cultura/economia , Meios de Cultura/metabolismo , Gluconacetobacter/crescimento & desenvolvimento , Etanol/metabolismoRESUMO
TonB-dependent receptors in concert with the TonB-ExbB-ExbD protein complex are responsible for the uptake of iron and substances such as vitamin B12 in several bacterial species. In this study, Tn5 mutagenesis of the sugarcane endophytic bacterium Gluconacetobacter diazotrophicus led to the isolation of a mutant with a single Tn5-insertion in the promoter region of a tonB gene ortholog. This mutant, named Gdiaa31, displayed a reduced growth rate and a lack of response to iron availability when compared to the wild-type strain PAL5(T). Several efforts to generate null-mutants for the tonB gene by insertional mutagenesis were without success. RT-qPCR analysis demonstrated reduced transcription of tonB in Gdiaa31 when compared to PAL5(T). tonB transcription was inhibited in the presence of Fe(3+) ions both in PAL5(T) and in Gdiaa31. In comparison with PAL5(T), Gdiaa31 also demonstrated decreased nitrogenase activity and biofilm formation capability, two iron-requiring physiological characteristics of G. diazotrophicus. Additionally, Gdiaa31 accumulated higher siderophore levels in culture supernatant. The genetic complementation of the Gdiaa31 strain with a plasmid that carried the tonB gene including its putative promoter region (pP(tonB)) restored nitrogenase activity and siderophore accumulation phenotypes. These results indicate that the TonB complex has a role in iron/siderophore transport and may be essential in the physiology of G. diazotrophicus.
Assuntos
Proteínas de Bactérias/genética , Gluconacetobacter/genética , Proteínas de Membrana/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Sideróforos/genética , Transporte Biológico/genética , Meios de Cultura/química , Teste de Complementação Genética , Gluconacetobacter/enzimologia , Gluconacetobacter/metabolismo , Ferro/metabolismo , Mutagênese Insercional , Mutação , Nitrogenase/genética , Fenótipo , Sideróforos/análise , Sideróforos/metabolismoRESUMO
Chitosan films reinforced with bacterial cellulose (BC) nanoribbons were studied to understand the influence of acid (acetic and lactic acids) on the reinforcing effect. For both acids, the maximum concentration of the reinforcing constituent was 5wt% with respect to the dry weight of chitosan. The infrared spectra, mechanical properties, morphology and antimicrobial activity of the films were analyzed. The results showed a difference between the acids in their behavior and effect on the reinforcement, with a tensile strength of 12.3MPa for the acetic acid films and 3.3MPa for the lactic acid films. Additionally, the bacterial inhibition tests were shown to be positive for the lactic acid films and negative for the acetic acid films. Therefore, exchanging the acid used in these films may be desirable for certain applications.
Assuntos
Ácido Acético/química , Celulose/biossíntese , Celulose/química , Quitosana/química , Gluconacetobacter/metabolismo , Ácido Láctico/química , Nanoestruturas/química , Embalagem de AlimentosRESUMO
Bacterial cellulose (BC) produced by Gluconacetobacter hansenii is a suitable biopolymer for biomedical applications. In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions. SEM images showed effects on fiber density and porosity on both sides of the BC membranes. Both enriched media decreased the BET surface area, water holding capacity, and rehydration rate. Fourier transform infrared (attenuated total reflectance mode) spectroscopy (FTIR-ATR) analysis revealed no change in the chemical structure of BC. L929 fibroblast cells were seeded on all BC-based membranes and evaluated in aspects of cell adhesion, proliferation and morphology. BCG1 membranes showed the highest biological performance and hold promise for the use in tissue engineering applications.
Assuntos
Celulose/química , Meios de Cultura/química , Dextrinas/química , Glucose/química , Manitol/química , Membranas Artificiais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Celulose/metabolismo , Meios de Cultura/farmacologia , Fibroblastos/citologia , Gluconacetobacter/efeitos dos fármacos , Gluconacetobacter/crescimento & desenvolvimento , Gluconacetobacter/metabolismo , Humanos , PorosidadeRESUMO
The present study was conducted to evaluate the cellulose production by Gluconacetobacter kakiaceti GM5 by means of two aerobic treatments: the static discontinuous fermentation process (treatment 1) and the discontinuous fermentation process in a rotary shaker (treatment 2). All these experiments were carried out using vinasse as experimental culture media (VM) and were compared with standard media containing glucose at 2% (standard medium (SM)). A sample of each treatment was extracted every 24 h over a period of 168 h. The maximum rates of cellulose produced in treatment 1 using SM added up to 3.63 ± 0.18 g l(-1), and to 4.15 ± 0.16 g l(-1) when VM was used. The amount of cellulose produced in treatment 2 using SM was 2.95 ± 0.09 g l(-1) (which suggests an increase of 37%), and added up to 1.84 ± 0.07 g l(-1) when using VM. A better global yield of both treatments in terms of sugar consumption after 168 h was obtained when using VM: 32% in treatment 1, whereas in treatment 2 it was 9%. A 20% decrease on vinasse COD (Chemical Oxygen Demand) values was found to be yet another important advantage of working with this strain.
Assuntos
Celulose/metabolismo , Meios de Cultura/química , Gluconacetobacter/metabolismo , AerobioseRESUMO
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium that has been found colonizing several plants. This acid-tolerant bacterium produces phytohormones that promote plant growth and is also able to grow in high-sugar concentrations. It has been demonstrated that exopolysaccharides (EPS), which are produced by strain Pal5 of G. diazotrophicus, play an important role in plant infection. We have investigated the structure of the EPS, which was produced by a strain of Pal5 grown in liquid medium containing mannitol as the sole carbon source. The results reveal an EPS with Glc, Gal, Man in a molar ratio of 6:3:1, respectively. NMR spectroscopy and chemical derivatization have revealed that the EPS structure has 4-O-substituted units of ß-glucose, 3-O-substituted units of ß-galactose and 2-O-substituted units of α-mannose. Glucose and galactose units linked at C6 were also found. The structure proposed herein is different from EPS produced by other species of Gluconacetobacter published to date.
Assuntos
Gluconacetobacter/metabolismo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/química , Gluconacetobacter/fisiologia , Glicosilação , Hidrólise , Peso Molecular , Monossacarídeos/química , Plantas/microbiologiaRESUMO
Background: Growth of Gluconacetobacter diazotrophicus with glucose as carbon an energy source has been extensively studied. However, there are no reports in the literature describing growth of G. diazotrophicus in cultures containing sucrose as carbon source. The first step in sucrose pathway and production of levans was investigated. Biomass, levans, gluconic acid and keto gluconic acids production and levansucrase activity were determined in cultures with different sucrose concentration and nitrogen sources. Results: The biomass production was maximal in cultures containing 100 g x L-1 sucrose and inorganic nitrogen. Gluconic acid production was observed under all conditions tested, at levels up to 9 g x L-1 in cultures with sucrose excess and biological N2-fixation (BNF). Keto gluconic acids were detectable only in cultures with sucrose excess and supplemented with organic nitrogen sources. Levans production, although observed in all cultures, was maximal in batch culture with 100 g x L-1 of sucrose and BNF, concomitant with a significant expression of extracellular levansucrase. Conclusions: Ours results not only describe some unknown aspects of G. diazotrophicus physiology, but open up the possibility of developing a technology of levans production by this organism using culture media with sucrose (or some cheaper substitute, like molasses) and without the addition of any N-source because of its ability of fixing atmospheric N2.
Assuntos
Gluconacetobacter/metabolismo , Frutanos/metabolismo , Sacarose/metabolismo , Cromatografia Líquida de Alta Pressão , Biomassa , Gluconacetobacter/crescimento & desenvolvimento , Técnicas de Cultura Celular por Lotes , Frutanos/análiseRESUMO
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.
Assuntos
Proteínas de Bactérias/genética , Gluconacetobacter/genética , Ferro/metabolismo , Óperon , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Transporte Biológico , Gluconacetobacter/metabolismo , Proteínas de Membrana Transportadoras/genética , Fixação de Nitrogênio , Proteínas Periplásmicas de Ligação/genética , Saccharum/microbiologiaRESUMO
The mechanisms of cadmium, cobalt and zinc resistance were characterized in the plant-growth-promoting bacterium Gluconacetobacter diazotrophicus PAl 5. The resistance level of the wild-type strain was evaluated through the establishment of minimum inhibitory concentrations (MIC) of the soluble compounds CdCl2·H2O, CoCl2·6H2O and ZnCl2. Gluconacetobacter diazotrophicus PAl 5 was resistant to high concentrations of Cd, Co and Zn, with MICs of 1.2, 20 and 20 mM, respectively. Screening of an insertion library from transposon EZ-Tn5
Assuntos
Cádmio/toxicidade , Cobalto/toxicidade , Gluconacetobacter/efeitos dos fármacos , Zinco/toxicidade , Sequência de Aminoácidos , Southern Blotting , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese Insercional , FilogeniaRESUMO
Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.
Assuntos
Carbono/metabolismo , Perfilação da Expressão Gênica/métodos , Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Perfilação da Expressão Gênica/normas , Genes Bacterianos , Gluconacetobacter/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/normasRESUMO
The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.
Assuntos
Acil-Butirolactonas/química , Acil-Butirolactonas/metabolismo , Gluconacetobacter/química , Gluconacetobacter/metabolismo , Acil-Butirolactonas/isolamento & purificação , Cromatografia Líquida , Meios de Cultura/química , Meios de Cultura/metabolismo , Espectrometria de MassasRESUMO
A bacterial strain isolated from the fermentation of Colombian homemade vinegar, Gluconacetobacter medellensis, was investigated as a new source of bacterial cellulose (BC). The BC produced from substrate media consisting of various carbon sources at different pH and incubation times was quantified. Hestrin-Schramm (HS) medium modified with glucose led to the highest BC yields followed by sucrose and fructose. Interestingly, the microorganisms are highly tolerant to low pH: an optimum yield of 4.5 g/L was achieved at pH 3.5, which is generally too low for other bacterial species to function. The cellulose microfibrils produced by the new strain were characterized by scanning and transmission electron microscopy, infrared spectroscopy X-ray diffraction and elemental analysis. The morphological, structural and chemical characteristics of the cellulose produced are similar to those expected for BC.
Assuntos
Celulose/biossíntese , Gluconacetobacter/metabolismo , Glucose/metabolismo , Celulose/químicaRESUMO
The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.
Assuntos
Biofilmes/crescimento & desenvolvimento , Genes Bacterianos/genética , Gluconacetobacter/metabolismo , Oryza/microbiologia , Polissacarídeos Bacterianos/metabolismo , Endófitos , Teste de Complementação Genética , Genoma Bacteriano/genética , Gluconacetobacter/genética , Gluconacetobacter/fisiologia , Proteínas de Fluorescência Verde , Hidroponia , Família Multigênica , Mutagênese Insercional , Fixação de Nitrogênio , Raízes de Plantas/microbiologia , Polissacarídeos Bacterianos/isolamento & purificação , Plântula/microbiologia , SimbioseRESUMO
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.
Assuntos
Proteínas de Bactérias/análise , Gluconacetobacter/metabolismo , Proteoma/análise , Saccharum/microbiologia , Simbiose/fisiologia , Adaptação Fisiológica , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Regulação Bacteriana da Expressão Gênica , Genótipo , Gluconacetobacter/genética , Gluconacetobacter/fisiologia , Fixação de Nitrogênio/genética , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismo , Proteoma/fisiologia , Saccharum/genética , Saccharum/crescimento & desenvolvimento , Saccharum/metabolismo , Transdução de SinaisRESUMO
Gluconacetobacter diazotrophicus is a micro-aerobic bacterium able to fix atmospheric nitrogen in endophytic mode. A proteomic approach was used to analyze proteins differentially expressed in the presence and absence of sugarcane plantlets. Two-dimensional gel electrophoresis (2-DE) showed 42 spots with altered levels of expression. Analysis of these spots by matrix-assisted laser desorption ionization time-of-flight in tandem (MALDI-TOF-TOF) identified 38 proteins. Differentially expressed proteins were associated with carbohydrate and energy metabolism, folding, sorting and degradation processes, and transcription and translation. Among proteins expressed in co-cultivated bacteria, four belong to membrane systems; others, like a transcription elongation factor (GreA), a 60 kDa chaperonin (GroEL), and an outer membrane lipoprotein (Omp16) have also been described in other plant-bacteria associations, indicating a common protein expression pattern as a result of symbiosis. A high protein content of 60kDa chaperonin isoforms was detected as non-differentially expressed proteins of the bacteria proteome. These results allow the assessment of the physiological significance of specific proteins to G. diazotrophicus metabolism and to the pathways involved in bacteria-host endophytic interaction.
Assuntos
Proteínas de Bactérias/análise , Gluconacetobacter/genética , Interações Hospedeiro-Patógeno/genética , Proteoma/análise , Saccharum/microbiologia , Metabolismo dos Carboidratos/genética , Técnicas de Cocultura , Metabolismo Energético/genética , Gluconacetobacter/metabolismo , Simbiose/genéticaRESUMO
BACKGROUND: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. RESULTS: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. CONCLUSION: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.