M. B. R. Steffens

Phenotypic and genotypic traits of Shiga toxin-producing Escherichia coli strains isolated from beef cattle from Paraná State, southern Brazil

Aims:  To investigate the prevalence and characteristics of Shiga toxin‐producing Escherichia coli (STEC) in cattle from Paraná State, southern Brazil.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

Structural organization and regulation of the nif genes of Herbaspirillum seropedicae

Abstract The promoter regions of nifA and nifH genes of Herbaspirillom seropedicae have been isolated and investigated, and the nifA promoter has been characterized in detail. Both regions contain a −24 −12 type promoter and NifA-binding sites; the nifA promoter also has an NtrC-binding site. The nifA expression is activated by NtrC and repressed by ammonium ions but not by oxygen. When the NtrC-binding site was deleted nifA expression was NifA-dependent. The native NifA protein of H. seropedicae was unable to activate nifH expression in Escherichia coli whereas a truncated protein, lacking the N-terminal domain was able to activate nifH expression in the absence of oxygen and presence of iron, indicating that the N-terminal domain regulates NifA activity. The truncated protein also activates nifH in Azospirillum brasilense in the presence or absence of ammonium ions. This suggests that the N-terminal domain senses the nitrogen status of the cell.

FGAP: an automated gap closing tool

BackgroundThe fast reduction of prices of DNA sequencing allowed rapid accumulation of genome data. However, the process of obtaining complete genome sequences is still very time consuming and labor demanding. In addition, data produced from various sequencing technologies or alternative assemblies remain underexplored to improve assembly of incomplete genome sequences.FindingsWe have developed FGAP, a tool for closing gaps of draft genome sequences that takes advantage of different datasets. FGAP uses BLAST to align multiple contigs against a draft genome assembly aiming to find sequences that overlap gaps. The algorithm selects the best sequence to fill and eliminate the gap.ConclusionsFGAP reduced the number of gaps by 78% in an E. coli draft genome assembly using two different sequencing technologies, Illumina and 454. Using PacBio long reads, 98% of gaps were solved. In human chromosome 14 assemblies, FGAP reduced the number of gaps by 35%. All the inserted sequences were validated with a reference genome using QUAST. The source code and a web tool are available at http://www.bioinfo.ufpr.br/fgap/.

A prospective study on Shiga toxin‐producing Escherichia coli in children with diarrhoea in Paraná State, Brazil

Aims:  To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC.

Proteomic analysis of Herbaspirillum seropedicae reveals ammonium-induced AmtB-dependent membrane sequestration of PII proteins

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

Role of PII proteins in nitrogen fixation control of Herbaspirillum seropedicae strain SmR1

BackgroundThe PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In Herbaspirillum seropedicae, two PII-like proteins (GlnB and GlnK), encoded by the genes glnB and glnK, were identified. The glnB gene is monocistronic and its expression is constitutive, while glnK is located in the nlmAglnKamtB operon and is expressed under nitrogen-limiting conditions.ResultsIn order to determine the involvement of the H. seropedicae glnB and glnK gene products in nitrogen fixation, a series of mutant strains were constructed and characterized. The glnK- mutants were deficient in nitrogen fixation and they were complemented by plasmids expressing the GlnK protein or an N-truncated form of NifA. The nitrogenase post-translational control by ammonium was studied and the results showed that the glnK mutant is partially defective in nitrogenase inactivation upon addition of ammonium while the glnB mutant has a wild-type phenotype.ConclusionsOur results indicate that GlnK is mainly responsible for NifA activity regulation and ammonium-dependent post-translational regulation of nitrogenase in H. seropedicae.

Prokaryotic communities of acidic peatlands from the southern Brazilian Atlantic Forest

The acidic peatlands of southern Brazil are ecosystems essential for the maintenance of the Atlantic Forest, one of the 25 hot-spots of biodiversity in the world. In this work, we investigated the composition of prokaryotic communities in four histosols of three acidic peatland regions by constructing small-subunit (SSU) rRNA gene libraries and sequencing. SSU rRNA gene sequence analysis showed the prevalence of Acidobacteria (38.8%) and Proteobacteria (27.4%) of the Bacteria domain and Miscellaneous (58%) and Terrestrial (24%) groups of Crenarchaeota of the Archaea domain. As observed in other ecosystems, archaeal communities showed lower richness than bacterial communities. We also found a limited number of Euryarchaeota and of known methanotrophic bacteria in the clone libraries.

Identification and characterization of PhbF: A DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1

BackgroundHerbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism.ResultsIn this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes.ConclusionsOur results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organisms resistance and survival in planta.

Polymorphisms of the promoter and exon 3 of the receptor for advanced glycation end products (RAGE) in Euro- and Afro-Brazilians

The receptor for advanced glycation end products (RAGE or AGER), a member of the immunoglobulin superfamily, is involved in pathologies such as atherosclerosis and diabetes. Over 50 SNPs were reported for RAGE, among which were the promoter region polymorphisms −429T>C (rs1800625), −374T>A (rs1800624) and a 63‐bp deletion (−407 to −345 bp), all related to increased RAGE expression. Additionally, in the exon 3, a putative site of binding ligands, the missense variation G82S (rs2070600) was associated with skin disorders in patients with diabetes. We have determined allele, genotype and haplotype frequencies of RAGE polymorphisms −429T>C, −374T>A, 63‐bp deletion and G82S in Euro‐Brazilians (n = 108) and Afro‐Brazilians (n = 91), characterized according to the predominant ancestry of the individuals. The allele frequencies for Euro‐ and Afro‐Brazilians were as follows: −429C, 12.5% vs. 12.1% (P = 0.90); −374A, 31.5% vs. 26.2% (P = 0.25); 63del, 0.0% vs. 3.8% (P = 0.004); and 82S, 1.9% vs. 0.6% (P = 0.24). Absolute linkage disequilibrium was found between the promoter polymorphisms −429T>C and −374T>A plus the 63‐bp deletion (D′=1.000; P < 0.0001). The haplotype frequencies differed (P = 0.003) between Euro‐ and Afro‐Brazilians. Our results showed that the frequencies of the 63‐bp deletion were higher in Afro‐Brazilians, while the other analysed polymorphisms were similarly distributed in the studied populations. The −374T>A plus 63‐bp deletion polymorphism captures more than 80% of the haplotypic variation in the studied population.