Eliane Aparecida Gomes

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) widespread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene.

Aim:  To avoid the limitations of 16S rRNA‐based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta‐subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies.

Polymorphism in the internal transcribed spacer (ITS) of the ribosomal DNA of 26 isolates of ectomycorrhizal fungi

Inter- and intraspecific variation among 26 isolates of ectomycorrhizal fungi belonging to 8 genera and 19 species were evaluated by analysis of the internal transcribed sequence (ITS) of the rDNA region using restriction fragment length polymorphism (RFLP). The ITS region was first amplified by polymerase chain reaction (PCR) with specific primers and then cleaved with different restriction enzymes. Amplification products, which ranged between 560 and 750 base pairs (bp), were obtained for all the isolates analyzed. The degree of polymorphism observed did not allow proper identification of most of the isolates. Cleavage of amplified fragments with the restriction enzymes Alu I, Hae III, Hinf I, and Hpa II revealed extensive polymorphism. All eight genera and most species presented specific restriction patterns. Species not identifiable by a specific pattern belonged to two genera: Rhizopogon (R. nigrescens, R. reaii, R. roseolus, R. rubescens and Rhizopogon sp.), and Laccaria (L. bicolor and L. amethystea). Our data confirm the potential of ITS region PCR-RFLP for the molecular characterization of ectomycorrhizal fungi and their identification and monitoring in artificial inoculation programs.

Molecular characterization and evaluation of pectinase and cellulase production of Penicillium spp.

Penicillium species were analyzed with molecular markers and for pectinase and cellulase production. RAPD and PCR-RFLP analysis indicated high polymorphism among at least 5 of 10 Penicillium species. Five species were chosen for pectinase and cellulase production in liquid medium and four of which appeared similar based on molecular analyses. P. brevicompactum and P. griseoroseum gave the highest pectinase production and were highly divergent by molecular techniques.

Auxin production and detection of the gene coding for the Auxin Efflux Carrier (AEC) protein in Paenibacillus polymyxa.

Different species of Paenibacillus are considered to be plant growth-promoting rhizobacteria (PGPR) due to their ability to repress soil borne pathogens, fix atmospheric nitrogen, induce plant resistance to diseases and/or produce plant growth-regulating substances such as auxins. Although it is known that indole-3-acetic acid (IAA) is the primary naturally occurring auxin excreted by Paenibacillus species, its transport mechanisms (auxin efflux carriers) have not yet been characterized. In this study, the auxin production of P. polymyxa and P. graminis, which are prevalent in the rhizospheres of maize and sorghum sown in Brazil, was evaluated. In addition, the gene encoding the Auxin Efflux Carrier (AEC) protein from P. polymyxa DSM36T was sequenced and used to determine if various strains of P. polymyxa and P. graminis possessed this gene. Each of the 68 P. polymyxa strains evaluated in this study was able to produce IAA, which was produced at concentrations varying from 1 to 17 μg/ml. However, auxin production was not detected in any of the 13 P. graminis strains tested in this study. Different primers were designed for the PCR amplification of the gene coding for the AEC in P. polymyxa, and the predicted protein of 319 aa was homologous to AEC from Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis. However, no product was observed when these primers were used to amplify the genomic DNA of seven strains of P. graminis, which suggests that this gene is not present in this species. Moreover, none of the P. graminis genomes tested were homologous to the gene coding for AEC, whereas all of the P. polymyxa genomes evaluated were. This is the first study to demonstrate that the AEC protein is present in P. polymyxa genome.

Molecular analysis of endophytic bacteria from the genus Bacillus isolated from tropical maize (Zea mays L.)

Endophytic bacteria play an important role in agriculture by improving plant performance and adaptation against biotic and abiotic stresses. In the present study molecular methods were used for identifying Bacillus endophytic bacteria isolated from Brazilian sweet corn. SDS-PAGE of wholecell protein extract of fortytwo isolates revealed a high number of scrutinable bands. Twenty-four isolates were identified in nine different groups of duplicated bacteria and eighteen were identified as unique. Some high-accumulated polipeptides with variable length were observed in almost isolates. Partial sequencing of 16S ribosomal gene revealed that all isolates are Bacillus sp. and among thirteen isolates with similar protein profiles, two were different strains. Among the forty-two isolates identified by rDNA sequencing, Bacillus subitilis and B. pumilus were the most frequenty species (15 and 12 isolates, respectively) followed by B. licheniformes (7 isolates), B. cereus (5 isolates) and B. amiloliquefascens (3 isolates). According to present results, SDS-PAGE technique could be used as a fast and cheap first tool for identifying interspecific variation in maize endophytic bacterial collections while rDNA sequencing could be applied for analyzing intraspecific variation among isolates with similar protein profile as well as for taxonomic studies.

Genetic diversity of indigenous common bean (Phaseolus vulgaris L.) rhizobia from the state of Minas Gerais, Brazil

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

In Vivo Insertional Mutagenesis in Corynebacterium pseudotuberculosis: an Efficient Means To Identify DNA Sequences Encoding Exported Proteins

ABSTRACT The reporter transposon-based system TnFuZ was used to identify exported proteins of the animal pathogen Corynebacterium pseudotuberculosis. Thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity. This activity was from 21 C. pseudotuberculosis loci that code for fimbrial and transport subunits and for hypothetical and unknown-function proteins.

Temporal dynamics of microbial communities in the rhizosphere of two genetically modified (GM) maize hybrids in tropical agrosystems

The use of genetically modified (GM) plants still raises concerns about their environmental impact. The present study aimed to evaluate the possible effects of GM maize, in comparison to the parental line, on the structure and abundance of microbial communities in the rhizosphere. Moreover, the effect of soil type was addressed. For this purpose, the bacterial and fungal communities associated with the rhizosphere of GM plants were compared by culture-independent methodologies to the near-isogenic parental line. Two different soils and three stages of plant development in two different periods of the year were included. As evidenced by principal components analysis (PCA) of the PCR-DGGE profiles of evaluated community, clear differences occurred in these rhizosphere communities between soils and the periods of the year that maize was cultivated. However, there were no discernible effects of the GM lines as compared to the parental line. For all microbial communities evaluated, soil type and the period of the year that the maize was cultivated were the main factors that influenced their structures. No differences were observed in the abundances of total bacteria between the rhizospheres of GM and parental plant lines.

Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.