Nitrate and nitrite transport in Escherichia coli

2005 ◽  
Vol 33 (1) ◽  
pp. 159-161 ◽  
Author(s):  
W. Jia ◽  
J.A. Cole
Keyword(s):  
Escherichia Coli ◽  
Single Channel ◽  
Exponential Phase ◽  
Site Directed Mutagenesis ◽  
Nitrate Transport ◽  
Nitrite Uptake ◽  
Nitrite Transport ◽  
Nitrate And Nitrite ◽  
Polytopic Membrane Protein ◽  
Positively Charged

Two polytopic membrane proteins, NarK and NarU, are involved in nitrate and nitrite uptake and nitrite extrusion by Escherichia coli. A third polytopic membrane protein, NirC, functions only in nitrite transport. During exponential growth, the quantity of NarU in membrane fractions was <0.01% of the quantity of NarK. During the stationary phase of growth, the ratio of NarU to NarK increased to 0.1%. However, in the exponential phase of growth, the strain expressing only NarK transports and reduces nitrate and nitrite at a rate only slightly higher than that of the strain expressing only NarU, indicating that, in a NarK+ strain, the rate of nitrate reduction is not limited by the rate of nitrate transport. By measuring nitrate and nitrite transport abilities of strains expressing only narK or expressing both narK and nirC, we hypothesized that NarK might function as a primary nitrate–nitrite antiporter. After nitrate is imported by NarK and reduced to nitrite, some nitrite is expelled from the cell and then reimported for reduction to ammonia. Two highly conserved positively charged residues, Arg-87 and Arg-303 of NarU, were shown by site-directed mutagenesis to play a key role in anion transport. This result indicates that NarU might form a single channel for nitrate and nitrite transport.

A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

2008 ◽  
Vol 417 (1) ◽  
pp. 297-307 ◽  
Author(s):  
Wenjing Jia ◽  
Nicholas Tovell ◽  
Stephanie Clegg ◽  
Mark Trimmer ◽  
Jeffrey Cole
Keyword(s):  
Escherichia Coli ◽  
Nitrate Uptake ◽  
Single Channel ◽  
Nitrite Reduction ◽  
Site Directed Mutagenesis ◽  
Extracellular Medium ◽  
Isotope Tracer ◽  
Wide Range ◽  
Nitrite Uptake ◽  
Major Facilitator

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and β-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg87 and Arg303 in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC− strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate–nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.

scholarly journals NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in Klebsiella oxytoca(pneumoniae) M5al

1998 ◽  
Vol 180 (5) ◽  
pp. 1311-1322 ◽  
Author(s):  
Qitu Wu ◽  
Valley Stewart
Keyword(s):  
Binding Protein ◽  
Klebsiella Oxytoca ◽  
Nitrogen Sources ◽  
Nitrate Transport ◽  
Periplasmic Binding Protein ◽  
Sequence Comparisons ◽  
Abc Protein ◽  
Nitrite Transport ◽  
Dependent Transport ◽  
Nitrate And Nitrite

ABSTRACT Klebsiella oxytoca can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the nasFEDCBA operon. We report here the complete nasFED sequence. Sequence comparisons indicate that the nasFED genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal nasFED genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the nasFED genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the ΔnasF strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.

scholarly journals Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic Escherichia coli Is Essential for Binding of 987P Fimbriae to Sulfatide

1999 ◽  
Vol 67 (11) ◽  
pp. 5755-5761 ◽  
Author(s):  
Byung-Kwon Choi ◽  
Dieter M. Schifferli
Keyword(s):  
Escherichia Coli ◽  
Essential Role ◽  
Surface Display ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Enterotoxigenic Escherichia Coli ◽  
Wild Type ◽  
Binding Properties ◽  
Brush Borders ◽  
Positively Charged

ABSTRACT The FasG subunit of the 987P fimbriae of enterotoxigenic strains ofEscherichia coli was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since fasG null mutants are nonfimbriated. In this study, fasG was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in fourfasG mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.

scholarly journals Mechanism of Action of RNase T

2002 ◽  
Vol 277 (51) ◽  
pp. 50155-50159 ◽  
Author(s):  
Yuhong Zuo ◽  
Murray P. Deutscher
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Mechanism Of Action ◽  
Rna Processing ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Detailed Model ◽  
Catalytic Center ◽  
Conserved Sequence ◽  
Positively Charged

Escherichia coliRNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.

scholarly journals Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase

1992 ◽  
Vol 267 (32) ◽  
pp. 22830-22836 ◽  
Author(s):  
K Ostanin ◽  
E.H. Harms ◽  
P.E. Stevis ◽  
R Kuciel ◽  
M.M. Zhou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acid Phosphatase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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