Kátia Regina dos Santos Teixeira

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5

BackgroundGluconacetobacter 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.ResultsGluconacetobacter 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.ConclusionThe 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.

Isolation and characterization of diazotrophic bacteria from banana and pineapple plants

Banana and pineapple fruit crops are widely cultivated in tropical areas where high amounts of fertilizers are applied, principally nitrogen. Over 200 kg N.ha-1.yr-1 is often applied to these crops. Nevertheless, developing countries face the problem of high costs of chemical fertilizers. As already demonstrated for other tropical crops, like sugar cane, the utilization of nitrogen-fixing bacteria may support the growth of these fruit plants. In this work, we demonstrate the association of nitrogen-fixing bacteria with banana and pineapple. Samples from roots, stems, leaves and fruits of different genotypes showed the occurrence of diazotrophic bacteria, when evaluated in semi-specific semi-solid media. These isolates could be separated into seven different groups according to their morphological and physiological characteristics. Additional, phylogenetic assignments were performed with group- and species-specific oligonucleotide probes. Bacteria related to the groups of Azospirillum amazonense, Azospirillum lipoferum, Burkholderia sp. and a group similar to the genus Herbaspirillum could be detected in samples of both crops. However, Azospirillum brasilense and another two groups of Herbaspirillum-like bacteria were detected only in banana plants. Two isolates of the latter group were identified as Herbaspirillum seropedicae, whereas the other isolates may represent a new Herbaspirillum species.

Molecular dissimilarities of Rhipicephalus sanguineus (Acari: Ixodidae) in Brazil and its relation with samples throughout the world: is there a geographical pattern?

In this study the genetic variability of Rhipicephalus sanguineus within Brazil and its relation with ticks of the same group from different continents was evaluated. Mitochondrial 12S and 16S rDNA fragments of R. sanguineus from seven Brazilian States were sequenced and compared to GenBank sequences of R. sanguineus and R. turanicus ticks from Africa, Asia, Europe, South America and USA. Results indicate a relatively high intra-specific variability between Brazilian samples but also a global latitude linked distribution pattern of at least two major R. sanguineus groups; one group distributed from latitude 25°N to 22°S including R. sanguineus from Brazil, Taiwan and Thailand and R. turanicus from Zambia and Zimbabwe, and the other group found closer to the poles, roughly above 29°N and below 30°S with ticks from Argentina, Uruguay, France, Oklahoma (USA), Israel and Egypt.

Analysis of nif and regulatory genes in Acetobacter diazotrophicus

Abstract The focus of our collaborative research program is the identification and characterization of nif and related regulatory genes of Acetobacter diazotrophicus in order to understand what factors influence nitrogen fixation in this unique diazotroph. To date, the following genes have been isolated from genomic libraries and are being analyzed: nifHDK, nifA, nifB, nifV, nifE and ntrBC. In addition, Nif− mutants have been constructed by insertional mutagenesis. These mutants are currently being used in inoculation experiments of sterile sugarcane plants to determine whether nitrogen fixed by A. diazotrophicus is significant for plant nutrition.

Protein expression profile of Gluconacetobacter diazotrophicus PAL5, a sugarcane endophytic plant growth-promoting bacterium

This is the first broad proteomic description of Gluconacetobacter diazotrophicus, an endophytic bacterium, responsible for the major fraction of the atmospheric nitrogen fixed in sugarcane in tropical regions. Proteomic coverage of G. diazotrophicus PAL5 was obtained by two independent approaches: 2‐DE followed by MALDI‐TOF or TOF‐TOF MS and 1‐DE followed by chromatography in a C18 column online coupled to an ESI‐Q‐TOF or ESI‐IT mass spectrometer. The 583 identified proteins were sorted into functional categories and used to describe potential metabolic pathways for nucleotides, amino acids, carbohydrates, lipids, cofactors and energy production, according to the Enzyme Commission of Enzyme Nomenclature (EC) and Kyoto Encyclopedia of genes and genomes (KEGG) databases. The identification of such proteins and their possible insertion in conserved biochemical routes will allow comparisons between G. diazotrophicus and other bacterial species. Furthermore, the 88 proteins classified as conserved unknown or unknown constitute a potential target for functional genomic studies, aiming at the understanding of protein function and regulation of gene expression. The knowledge of metabolic fundamentals and coordination of these actions are crucial for the rational, safe and sustainable interference on crops. The entire dataset, including peptide sequence information, is available as Supporting Information and is the major contribution of this work.

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.

What Is Expected from the Genus Azospirillum as a Plant Growth-Promoting Bacteria?

Bacteria capable of stimulating plant growth are generally known as plant growth-promoting bacteria (PGPB). Among them are Azospirillum species that influence plant growth through different mechanisms. Azospirillum is a Gram-negative bacterium that belongs to the alphaproteobacteria phylum. On the basis of the newly discovered species (at present 15), it is present not only in a wide diversity of plants, including those of agronomic importance such as cereals, sugarcane; and forage grasses, but also in other non-Poaceae plant species. Due to the capacity for improving plant yield in agronomically important crops, Azospirillum possesses biotechnological application as inoculant or biofertilizer. Among the mechanisms involved in promoting plant growth are N2 fixation, P solubilization, phytohormone production (auxins, cytokinins, and gibberellins), increased nutrient uptake, enhanced stress resistance, vitamin production, siderophores, and biocontrol activity. Some of them, as well as their agricultural application, are discussed in this chapter.

Nitrogen fixing bacteria in the family Acetobacteraceae and their role in agriculture

For centuries, the Acetobacteraceae is known as a family that harbors many species of organisms of biotechnological importance for industry. Nonetheless, since 1988 representatives of this family have also been described as nitrogen fixing bacteria able to plant growth promotion by a variety of mechanisms. Nitrogen fixation is a biological process that guarantees that the atmospheric N2 is incorporated into organic matter by several bacterial groups. Most representatives of this group, also known as diazotrophic, are generally associated with soil rhizosphere of many plants and also establishing a more specific association living inside roots, leaves, and others plants tissues as endophyte. Their roles as plant growth‐promoting microorganisms are generally related to increase in plant biomass, phosphate and other mineral solubilization, and plant pathogen control. Here, we report many of these plant growth‐promoting processes related to nitrogen fixing species already described in Acetobacteraceae family, especially Gluconacetobacter diazotrophicus and their importance to agriculture. In addition, a brief review of the state of art of the phylogenetics, main physiological and biochemical characteristics, molecular and functional genomic data of this group of Acetobacteraceae is presented.

Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

Azospirillum is a plant growth‐promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non‐traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon‐source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols.

Analysis of nitrogen fixation and regulatory genes in the sugar cane endophyte Acetobacter diazotrophicus

The specific endophytic association between Acetobacter diazotrophicus and sugarcane may represent a beneficial symbiosis with the bacterial partner providing fixed N and/or plant growth promoting factors such as auxin. Our goals are to identify and characterize the genes of A. diazotrophicus necessary for nitrogen fixation and effective symbiosis. In summary, we have isolated a gene cluster including nifAB HDK ENXQ USVW, fixABCX in one region of the A. diazotrophicus genome. Also, two other genes, modD (molybdenum transporter protein) and mcpA (methyl-accepting chemotaxis protein) were located downstream of fixABCX. The mcpA gene product is involved in responses to extracellular chemotactic signals, which may play an important role in plant-microbe interactions. A mcpA mutant of A. diazotrophicus lost chemotaxis toward a wide range of attractant stimuli and will be used to determine whether mcpA + bacteria have a competitive advantage with respect to cell survival and colonization of sugarcane.