Use of lactose to induce expression of soluble NifA protein domains of Herbaspirillum seropedicae in Escherichia coli

2000 ◽  
Vol 46 (11) ◽  
pp. 1087-1090 ◽  
Author(s):  
Rose A. Monteiro ◽  
Emanuel M. Souza ◽  
M. Geoffrey Yates ◽  
Fábio O. Pedrosa ◽  
Leda S. Chubatsu
Keyword(s):  
Escherichia Coli ◽  
Protein Domains ◽  
Herbaspirillum Seropedicae ◽  
Nifa Protein

scholarly journals Fnr Is Involved in Oxygen Control of Herbaspirillum seropedicae N-Truncated NifA Protein Activity in Escherichia coli

2003 ◽  
Vol 69 (3) ◽  
pp. 1527-1531 ◽  
Author(s):  
Rose A. Monteiro ◽  
Emanuel M. de Souza ◽  
M. Geoffrey Yates ◽  
Fabio O. Pedrosa ◽  
Leda S. Chubatsu
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Ammonium Ions ◽  
Oxygen Control ◽  
Herbaspirillum Seropedicae ◽  
Protein Activity ◽  
Iron Deprivation ◽  
Iron Requirement ◽  
Fnr Protein ◽  
Nifa Protein

ABSTRACT Herbaspirillum seropedicae is an endophytic diazotroph belonging to the β-subclass of the class Proteobacteria, which colonizes many members of the Gramineae. The activity of the NifA protein, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through its N-terminal domain and by oxygen through mechanisms that are not well understood. Here we report that the NifA protein of H. seropedicae is inactive and more susceptible to degradation in an fnr Escherichia coli background. Both effects correlate with oxygen exposure and iron deprivation. Our results suggest that the oxygen sensitivity and iron requirement for H. seropedicae NifA activity involve the Fnr protein.

Use of lactose to induce expression of soluble NifA protein domains ofHerbaspirillum seropedicaeinEscherichia coli

2000 ◽  
Vol 46 (11) ◽  
pp. 1087-1090 ◽  
Author(s):  
Rose A Monteiro ◽  
Emanuel M Souza ◽  
M Geoffrey Yates ◽  
Fábio O Pedrosa ◽  
Leda S Chubatsu
Keyword(s):  
Escherichia Coli ◽  
Soluble Fraction ◽  
Herbaspirillum Seropedicae ◽  
Enhancer Binding Protein ◽  
Nifa Protein ◽  
Kinetics Of ◽  
Background Expression ◽  
Fix Genes

Overexpression and purification are procedures used to allow functional and structural characterization of proteins. Many overexpressed proteins are partially or completely insoluble, and can not be easily purified. The NifA protein is an enhancer-binding protein involved in activating the expression of nif and some fix genes. The NifA protein from many organisms is usually insoluble when over-expressed, and therefore difficult to work with in vitro. In this work we have overexpressed the central+C-terminal and the central domains of the Herbaspirrilum seropedicae NifA protein in an Escherichia coli background. Expression was induced with either IPTG or lactose. The data showed that induction with lactose promoted a significantly higher percentage of these proteins in the soluble fraction than with IPTG. This probably reflects a slower kinetics of induction by lactose.Key words: Herbaspirillum seropedicae, NifA protein, transcriptional activator, nitrogen fixation, protein expression.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein

2003 ◽  
Vol 27 (2) ◽  
pp. 313-318 ◽  
Author(s):  
Rose A Monteiro ◽  
Emanuel M Souza ◽  
M Geoffrey Yates ◽  
M.Berenice R Steffens ◽  
Fábio O Pedrosa ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Herbaspirillum Seropedicae ◽  
Terminal Domain ◽  
Nifa Protein

Cloning of a recA-like gene from the diazotroph Herbaspirillum seropedicae strain Z78

1993 ◽  
Vol 39 (12) ◽  
pp. 1096-1102 ◽  
Author(s):  
M. B. R. Steffens ◽  
L. U. Rigo ◽  
S. Funayama ◽  
E. M. Souza ◽  
H. B. Machado ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Reca Gene ◽  
Methyl Methanesulfonate ◽  
Herbaspirillum Seropedicae ◽  
E Coli ◽  
Tn5 Mutagenesis ◽  
Ultraviolet Resistance ◽  
Restriction Sites ◽  
Reca Mutant

A recombinant plasmid, pBMR5, carrying a recA-like gene of Herbaspirillum seropedicae, was isolated from a H. seropedicae genomic library by intergeneric complementation of Escherichia coli recA mutant strain HB101. Quantitative survival experiments showed that pBMR5 restored the ultraviolet radiation and methyl methanesulfonate resistances and recombinational proficiency of this strain. Hybridization studies showed that there is DNA sequence homology between the recA gene of E. coli K12 and that of H. seropedicae. Restriction sites for EcoRI, HindIII, BamHI, and BglII were found in the DNA insert derived from H. seropedicae in pBMR5. A Tn5 insertional mutant of pBMR5, called pBMR26.2, failed to restore recombination proficiency and methyl methanesulfonate and ultraviolet resistance to recA mutants of E. coli.Key words: Herbaspirillum seropedicae, nitrogen fixation, recA-like gene, Tn5 mutagenesis.

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