135 research outputs found
Clonagem e sequenciamento parcial do gene amtB de Azospirillum brasilense FP2
Orientadora: Leda Satie ChubastuMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciencias Biológicas. Curso de Graduaçao em Ciencias BiológicasResumo : Azospirillum brasilense é uma bactéria capaz de fixar nitrogênio (diazotrófica) que se associa com diversas gramíneas de interesse agrícola como milho, trigo e arroz. O transporte de amônia é de fundamental interesse em bactérias diazatrófi.cas devido a ação repressora deste íon sobre a fixação de nitrogênio. A nossa estratégia inicial para a clonagem do gene que codifica para o transportador de amônio (amtB) envolveu a amplificação de um pequeno fragmento deste gene por PCR, utilizando oligonudeotídeos sintetizados quimicamente. Para determinar a sequência dos oligonucleotídeos a serem utilizados nas amplificações foram comparadas sequências de aminoácidos, já, conhecidas, de transportadores de amônia de três organismos: Escherichia co/i, Mycobacterium tubercuJosis e Bacillus subtilis. As regiões que apresentavam alta homologia foram convertidas para sequência de nucleotídeos utilizando uma tabela de preferência de códons para A. brasilense. O fragmento de DNA ampHficado por PCR foi clonado e sequenciado (número de acesso no GenBank: AF082303). A sequência foi submetida a comparação com banca de dados e apresentou alta homologia com transportadores de íons amônia de diversos organismos. Este fragmento donado será utilizado como sonda em . hibridizações contra uma biblioteca genômica de, A. brasilense que estamos construindo para que consigamos clonar o gene completo
Evaluation of six methods for total dna extraction from gut earthworms for microbial community diversity and metagenomics.
Isolation and characterisation of metallothionein from the clam Ruditapes decussatus
Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus) using gel-permeation and ion-exchange chromatography. Four cadmium-metallothioneins (CdMTs) were resolved by ion-exchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd-S aggregates. The NH2-terminal sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH2-terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure
V79 Chinese-hamster cells rendered resistant to high cadmium concentration also become resistant to oxidative stress
Role of PII proteins in nitrogen fixation control of Herbaspirillum seropedicae strain SmR1
<p>Abstract</p> <p>Background</p> <p>The PII protein family comprises homotrimeric proteins which act as transducers of the cellular nitrogen and carbon status in prokaryotes and plants. In <it>Herbaspirillum seropedicae</it>, two PII-like proteins (GlnB and GlnK), encoded by the genes <it>glnB </it>and <it>glnK</it>, were identified. The <it>glnB </it>gene is monocistronic and its expression is constitutive, while <it>glnK </it>is located in the <it>nlmAglnKamtB </it>operon and is expressed under nitrogen-limiting conditions.</p> <p>Results</p> <p>In order to determine the involvement of the <it>H. seropedicae glnB </it>and <it>glnK </it>gene products in nitrogen fixation, a series of mutant strains were constructed and characterized. The <it>glnK<sup>- </sup></it>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 <it>glnK </it>mutant is partially defective in nitrogenase inactivation upon addition of ammonium while the <it>glnB </it>mutant has a wild-type phenotype.</p> <p>Conclusions</p> <p>Our results indicate that GlnK is mainly responsible for NifA activity regulation and ammonium-dependent post-translational regulation of nitrogenase in <it>H. seropedicae</it>.</p
Identification and characterization of PhbF: A DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1
<p>Abstract</p> <p>Background</p> <p><it>Herbaspirillum seropedicae </it>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.</p> <p>Results</p> <p>In this work we describe the characterization of the PhbF protein from <it>H. seropedicae </it>SmR1 which was purified and characterized after expression in <it>E. coli</it>. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in <it>H. seropedicae </it>SmR1. <it>In silico </it>analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using <it>lacZ </it>fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes.</p> <p>Conclusions</p> <p>Our results indicate that <it>H. seropedicae </it>SmR1 PhbF regulates expression of <it>phb</it>-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 organism's resistance and survival <it>in planta</it>.</p
The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecXHs) can interact with the H. seropedicae RecA protein (RecAHs) and that RecAHs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecXHs inhibited 90% of the RecAHs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecAHs. RecAHs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecXHs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecXHs protein negatively modulates the RecAHs activities by protein-protein interactions and also by DNA-protein interactions
Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes
Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)
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