PubMed Health⌕ Search

Biomedical subjects

R Cavicchioli

Publications and source records attributed to R Cavicchioli.

27 records · Page 2Linked to original sources
PubMed1995

The NarX and NarQ sensor-transmitter proteins of Escherichia coli each require two conserved histidines for nitrate-dependent signal transduction to NarL.

The NarX, NarQ, and NarL proteins of Escherichia coli constitute a two-component regulatory system that controls the expression of a number of anaerobic respiratory pathway genes in response to nitrate. NarX and NarQ are sensor-transmitter proteins that can independently detect the presence of nitrate in the cell environment and transmit this signal to the response regulator, NarL. Upon activation, NarL binds DNA and modulates the expression of its target genes by the repression or activation of transcription. NarX and NarQ each contain a conserved histidine residue that corresponds to the site of autophosphorylation of other sensor-transmitter proteins. They also contain a second conserved histidine residue that is present in the NarX, NarQ, UhpB, DegS, and ComP subfamily of sensor-transmitter proteins. The second histidine is located near a universally conserved asparagine residue, the role of which in signal transduction is unknown. To investigate the role of these conserved amino acids in the NarX and NarQ proteins, we mutated the narX and narQ genes by site-directed mutagenesis. In vivo, each mutation severely impaired NarL-dependent activation or repression of reporter gene expression in response to nitrate. The in vivo data suggest that the environmental signal nitrate controls both the kinase and phosphatase activities of the two sensor-transmitter proteins. The altered NarX and NarQ proteins were purified and shown to be defective in their ability to autophosphorylate in the presence of [gamma-32P]ATP. The NarX and NarQ proteins with amino acid substitutions at the first conserved histidine position were also unable to dephosphorylate NarL-phosphate in vitro. In contrast, the proteins containing amino acid substitutions at the second conserved histidine or at the conserved asparagine residue retained NarL-phosphate dephosphorylation activity. The conserved histidine and asparagine residues are essential for NarX and NarQ function, and this suggests that other two-component sensor-transmitter proteins may function in a similar fashion.

Amino Acid Sequence↗
PubMed1994

Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli.

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.

Bacterial Proteins↗
PubMed1992

Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli.

In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC), fumarate reductase (frdABCD) and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate-dependent control, and apparently function as members of a two-component regulatory system. NarX is a presumed sensor-transmitter for nitrate and possibly molybdenum detection. The presumed response-regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate-dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate-dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate-dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor-transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate-dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor-transmitter proteins.

Amino Acid Sequence↗
PubMed1991

The involvement of transcriptional read-through from internal promoters in the expression of a novel endoglucanase gene FSendA, from Fibrobacter succinogenes AR1.

Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1. Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1). The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1. Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression. Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination. The possibility of autogenous regulation by translation products is suggested.

Amino Acid Sequence↗
PubMed1991

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.

Bacteroides↗
PubMed1991

endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1.

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2.

Amino Acid Sequence↗
PubMed1986

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins.

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.

Heat-Shock Proteins↗
PubMed1984

Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast.

Heat shock acquisition of ethanol- and thermotolerance in Saccharomyces cerevisiae was not inhibited in cells incubated in the presence of cycloheximide or chloramphenicol. Respiratory-deficient (rho-) mutants also characteristically exhibited the heat shock response. It was concluded that mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast.

Chloramphenicol↗