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.     

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.     

Structural levansucrase gene (lsdA) constitutes a functional locus conserved in the species Gluconacetobacter diazotrophicus

Levansucrase (EC 2.4.1.10) was identified as a constitutive exoenzyme in 14 Gluconacetobacter diazotrophicus strains recovered from different host plants in diverse geographical regions. The enzyme, consisting of a single 60-kDa polypeptide, hydrolysed sucrose to synthesise oligofructans and levan. Sugar-cane-associated strains of the most abundant genotype (electrophoretic type 1) showed maximal values of levansucrase production. These values were three-fold higher than those of the isolates recovered from coffee plants. Restriction fragment length polymorphism analysis revealed a high degree of conservation of the levansucrase locus (IsdA) among the 14 strains under study, which represented 11 different G. diazotrophicus genotypes. Targeted disruption of the lsdA gene in four representative strains abolished their ability to grow on sucrose, indicating that the endophytic species G. diazotrophicus utilises plant sucrose via levansucrase.     

Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif- mutants strains

The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field by comparing plants inoculated with wild-type and Nif mutant MAd3A in two independent experiments. The wild-type and Nif mutant strains colonized sugarcane plants equally and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had a higher total N content 60 days after planting than did plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane might be a significant mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement was observed in plants inoculated with either wild-type or Nif- mutants, suggesting the additional effect of a plant growth promoting factor provided by A. diazotrophicus. A 15N2 incorporation experiment demonstrated that A. diazotrophicus wild-type strains actively fixed N2 inside sugarcane plants, whereas the Nif- mutants did not.     

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.     

Population Dynamics of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> in Sugarcane Cultivars and Its Effect on Plant Growth

Different experiments have estimated that the contribution of biological nitrogen fixation (BNF) is largely variable among sugarcane cultivars. Which bacteria are the most important in sugarcane-associated BNF is unknown. However, Gluconacetobacter diazotrophicus has been suggested as a strong candidate responsible for the BNF observed. In the present study, bacteria-free micropropagated plantlets of five sugarcane cultivars were inoculated with three G. diazotrophicus strains belonging to different genotypes. Bacterial colonization was monitored under different nitrogen fertilization levels and at different stages of plant growth. Analysis of the population dynamics of G. diazotrophicus strains in the different sugarcane varieties showed that the bacterial populations decreased drastically in relation to plant age, regardless of the nitrogen fertilization level, bacterial genotype or sugarcane cultivars. However, the persistence of the three strains was significantly longer in some cultivars (e.g., MEX 57-473) than in others (e.g., MY 55-14). In addition, some strains (e.g., PAl 5^T) persisted for longer periods in higher numbers than other strains (e.g., PAl 3) inside plants of all the cultivars tested. Indeed, the study showed that the inoculation of G. diazotrophicus may be beneficial for sugarcane plant growth, but this response is dependent both on the G. diazotrophicus genotype and the sugarcane variety. The most positive response to inoculation was observed with the combination of strain PAl 5^T and the variety MEX 57-473. Although the positive effect on sugarcane growth apparently occurred by mechanisms other than nitrogen fixation, the results show the importance of the sugarcane variety for the persistence of the plant–bacteria interaction, and it could explain the different rates of BNF estimated among sugarcane cultivars.     

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.     

Influence of carbon and nitrogen sources on growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus

The effects of different carbon and nitrogen sources on the growth, nitrogenase activity, and carbon metabolism of Gluconacetobacter diazotrophicus were investigated. The amino acids asparagine, aspartic acid, and glutamic acid affected microbial growth and nitrogenase activity. Several enzymatic activities involved in the tricarboxylic acid cycle were affected by the carbon source used. In addition, glucose and gluconate significantly increased the oxygen consumption (respiration rate) of whole cells of G. diazotrophicus grown under aerobic conditions. Enzymes responsible for direct oxidation of glucose and gluconate were especially active in cells grown with sucrose and gluconate. The presence of amino acids in the apoplastic and symplastic sap of sugarcane stems suggests that these compounds might be of importance in the regulation of growth and nitrogenase activity during the symbiotic association. The information obtained from the plant-bacterium association together with the results of other biochemical studies could contribute to the development of biotechnological applications of G. diazotrophicus.     

N-fertilizer saving by the inoculation of Gluconacetobacter diazotrophicus and Herbaspirillum sp. in micropropagated sugarcane plants

Colonization of micropropagated sugarcane plants by Gluconacetobacter diazotrophicus and Herbaspirillum sp. was confirmed by a dot-immunoblot assay. In all, a 45-day short-term and 180-day long-term experiments conducted on micropropagated sugarcane plants of Co 86032, a sugar rich popular variety in South India, indicated the usefulness of these diazotrophs as plant growth promoting bacteria. Co-inoculation of these two bacteria enhanced the biomass considerably under N-limited condition in the short duration experiment. In the long-term experiment, the establishment of inoculated Herbaspirillum sp. remained stable with the age of the crop up to 180 days, while there was a reduction in population of G. diazotrophicus for the same period. The total bio-mass and leaf N were higher in plants inoculated with G. diazotrophicus and Herbaspirillum sp. without N fertilization and also in plants with 50% of the recommended N (140 kg ha(-1)) than the plants fertilized with recommended dose of inorganic N (280 kg ha(-1)). This experiment showed that inoculation with these bacteria in sugarcane variety Co 86032 could mitigate fertilizer N application considerably in sugarcane cultivation.     

A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane

AIMS: To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers. METHODS AND RESULTS: Two N2-fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18. Both isolates showed acetylene reduction and H2 production in nitrogen-free media. Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037). The nifH gene segment was amplified from both isolates using specific primers. Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis. CONCLUSIONS: Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus. For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H2 production (up to 67.7 nmol H2 h(-1) 10(10) cell(-1)). Isolate T2 was a strain of Gluconacetobacter diazotrophicus. SIGNIFICANCE AND IMPACT OF THE STUDY: A new N2-fixing endophyte, i.e. Pantoea, able to produce H2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized. The strain with these attributes may well be valuable for agriculture.     

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.     

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media

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.     

Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus

AIMS: The effect of some abiotic factors, dryness, heat and salinity on the growth and biological activity of Gluconacetobacter diazotrophicus, and the influence of a salt stress on some enzymes involved in carbon metabolism of these bacteria is studied under laboratory conditions. METHODS AND RESULTS: Strain PAL-5 of G. diazotrophicus was incubated under different conditions of drying, heat and salinity. Cells showed tolerance to heat treatments and salt concentrations, and sensitivity to drying conditions. Higher NaCl dosage of 150 and 200 mmol l -1 limited its growth and drastically affected the nitrogenase activity and the enzymes glucose dehydrogenase, alcohol dehydrogenase, fumarase, isocitrate dehydrogenase and malate dehydrogenase. CONCLUSIONS: Gluconacetobacter diazotrophicus, despite its endophytic nature, tolerated heat treatments and salinity stress, but its nitrogenase activity and carbon metabolism enzymes were affected by high NaCl dosage. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the biological activity of G. diazotrophicus in response to different abiotic factors led to more knowledge of this endophyte and may help to clarify pathways involved in its transmission into the host plant.     

Presence of endophytic diazotrophs in sugarcane juice

Summary In this work we investigated the diazotrophs present in the juice of different varieties of sugarcane. Samples of the aerial part of sugarcane, between 3 and 5 months old, were assessed for the presence of diazotrophs. The isolated nitrogen-fixing microorganisms were identified as Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Azospirillum brasilense. These microorganisms use organic acids found in the sugarcane juice as an energy source. They also use sugars, such as free glucose, that are present in all the organs of the aerial part. The distribution of endophytic microorganisms in sugarcane depends on the different organic acid and sugar concentrations present during development and growth. We concluded that in more mature regions of the sugarcane stem Gluconacetobacter diazotrophicus grows more abundantly than Herbaspirillum seropedicae or Azospirillum brasilense.     

甘蔗赤腐病内生拮抗菌YC89的筛选及鉴定

【目的】筛选防治甘蔗赤腐病(sugarcane red rot)的生防菌株。【方法】实验以前期分离获得的甘蔗内生细菌为目标菌,以甘蔗赤腐病的病原真菌镰孢炭疽菌(Colletotrichum falcatum Went.)为指示菌,采用平板对峙法筛选对该病菌有较强抑制作用的菌株,然后通过琼脂扩散法测定菌株代谢产物对抑菌活性的影响,并对具有较好拮抗效果的高效菌株进行抑菌广谱性分析并对其进行鉴定。最后通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对高效菌株YC89进行鉴定。【结果】经初筛筛选到抑菌带均大于1.60 cm的5株拮抗细菌,其中X22、W2、YC89抑菌带均高达1.87 cm。对初筛得到的5株内生菌进行复筛,结果所示菌株YC89、H1、X22、W2、YT93对镰孢炭疽菌的抑菌率都在75%以上,其中菌株YC89对该病菌的抑菌效果最好,其抑菌率为78%。菌株YC89的发酵液、上清液、过滤液及粗蛋白提取液对镰孢炭疽菌的生长有较强的抑制作用,且菌株YC89对玉米大斑病、甘蔗梢腐病、草莓灰霉病等7种病原菌也有较好的抑制效果。通过菌株鉴定结果,初步将YC89菌株鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。【结论】菌株YC89对镰孢炭疽菌具有较好的抑制效果,表明其对甘蔗赤腐病的生物防治具有较大的应用潜力。     

Low recovery frequency of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> from plants and associated mealybugs in Cuban sugarcane fields

This study was aimed to isolate and identify the N₂-fixing bacterium Gluconacetobacter diazotrophicus from 11 sugarcane varieties, grown under field conditions in four Cuban provinces, and from their associated mealybugs Saccharicoccus sacchari. Identification was based on morphological and biochemical tests and PCR-amplification of 16S rRNA genes using species-specific primers. From all sugarcane varieties and numerous mealybug colonies sampled, G. diazotrophicus isolates were recovered from inside sugarcane stems of only three varieties, and one from S. sacchari colony. These four isolates showed acetylene reduction activity in nitrogen-free media and contained nifH genes which were PCR-amplified using specific primers. ERIC-PCR fingerprinting was used to compare the Cuban G. diazotrophicus isolates with type and reference strains of N₂-fixing Gluconacetobacteria. The very low frequency of G. diazotrophicus isolates recovered is probably related with the high doses of nitrogen fertilizers applied to the sugarcane in the Cuban fields for almost 30 years. Some genetic differences, using ERIC-PCR, were detected among G. diazotrophicus strains, which could be related with its source.     

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.