Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin.

The inducibility of hepatic cytosolic glutathione S-transferases (GSTs) was examined in brown bullheads, a freshwater fish that is highly susceptible to hepatic neoplasia following exposure to carcinogen-contaminated sediments. Juvenile bullheads were fed a semi-purified antioxidant-free diet supplemented with ethoxyquin (0.5% w/w dissolved in 3% corn oil), a prototypical rodent GST-inducing agent, twice daily for 14 days. Control bullheads received the antioxidant-free diet supplemented with corn oil (3% w/w). A significant increase (1.6-fold, p < or = 0.01) in hepatic cytosolic GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the ethoxyquin-treated bullheads relative to control fish. A trend toward increased GST-NBC activity was observed in the ethoxyquin-treated fish (1.2-fold, p = 0.06), whereas no treatment-related effects were observed on GST activities toward ethacrynic acid (ECA). In contrast, GST activity toward (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was repressed in affinity-purified cytosolic fractions prepared from ethoxyquin-treated bullheads relative to control bullheads. Silver staining and densitometric analysis of isoelectric-focused, affinity-purified GST proteins revealed increased expression of two basic GST-like isoforms in ethoxyquin-treated fish. In summary, exposure to ethoxyquin increases brown bullhead GST-CDNB catalytic activity and hepatic cationic GST protein expression. However, the increase in overall GST-CDNB activity by ethoxyquin is associated with repression of GST-BPDE activity, suggesting differential effects on hepatic bullhead GST isoforms by ethoxyquin. The potential repression of bullhead GST isoforms that conjugate the carcinogenic metabolites of PAH metabolism under conditions of environmental chemical exposure could be a contributing factor in the sensitivity of bullheads to pollutant-associated neoplasia.

Role of the RNA polymerase alpha subunits in MetR-dependent activation of metE and metH: important residues in the C-terminal domain and orientation requirements within RNA polymerase.

Many transcription factors activate by directly interacting with RNA polymerase (RNAP). The C terminus of the RNAP alpha subunit (alphaCTD) is a common target of activators. We used both random mutagenesis and alanine scanning to identify alphaCTD residues that are crucial for MetR-dependent activation of metE and metH. We found that these residues localize to two distinct faces of the alphaCTD. The first is a complex surface consisting of residues important for alpha-DNA interactions, activation of both genes (residues 263, 293, and 320), and activation of either metE only (residues 260, 276, 302, 306, 309, and 322) or metH only (residues 258, 264, 290, 294, and 295). The second is a distinct cluster of residues important for metE activation only (residues 285, 289, 313, and 314). We propose that a difference in the location of the MetR binding site for activation at these two promoters accounts for the differences in the residues of alpha required for MetR-dependent activation. We have designed an in vitro reconstitution-purification protocol that allows us to specifically orient wild-type or mutant alpha subunits to either the beta-associated or the beta'-associated position within RNAP (comprising alpha(2), beta, beta', and sigma subunits). In vitro transcriptions using oriented alpha RNAP indicate that a single alphaCTD on either the beta- or the beta'-associated alpha subunit is sufficient for MetR activation of metE, while MetR interacts preferentially with the alphaCTD on the beta-associated alpha subunit at metH. We propose that the different alphaCTD requirements at these two promoters are due to a combination of the difference in the location of the activation site and limits on the rotational flexibility of the alphaCTD.

The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli.

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic-binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA-phoA and dppA-lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

GcvA-mediated activation of gcvT-lacZ expression involves the carboxy-terminal domain of the alpha subunit of RNA polymerase.

Several LysR-type transcriptional regulators have been shown to require the carboxy-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase to activate their target genes. We show here that GcvA, a LysR-type protein, also uses the alphaCTD to activate the Escherichia coli gcvTHP operon. Amino acid residues in the alphaCTD important for GcvA-dependent activation, however, have no effect on GcvA-mediated repression of the operon.

The cyclic AMP receptor protein is dependent on GcvA for regulation of the gcv operon.

The Escherichia coli gcv operon is transcriptionally regulated by the GcvA, GcvR, Lrp, and PurR proteins. In this study, the cyclic AMP (cAMP) receptor protein (CRP) is shown to be involved in positive regulation of the gcv operon. A crp deletion reduced expression of a gcvT-lacZ fusion almost fourfold in glucose minimal (GM) medium. The phenotype was complemented by both the wild-type crp gene and four crp alleles that encode proteins with amino acid substitutions in known activating regions of CRP. A cyaA deletion also resulted in a fourfold decrease in gcvT-lacZ expression, and wild-type expression was restored by the addition of cAMP to the growth medium. A cyaA crp double deletion resulted in levels of gcvT-lacZ expression identical to those observed with either single mutation, showing that CRP and cAMP regulate through the same mechanism. Growth in GM medium plus cAMP or glycerol minimal medium did not result in a significant increase in gcvT-lacZ expression. Thus, the level of cAMP present in GM medium appears to be sufficient for regulation by CRP. DNase I footprint analysis showed that CRP binds and protects two sites centered at bp -313 (site 1) and bp -140 (site 2) relative to the transcription initiation site, but a mutational analysis demonstrated that only site 1 is required for CRP-mediated regulation of gcvT-lacZ expression. Expression of the gcvT-lacZ fusion in a crp gcvA double mutant suggested that CRP's role is dependent on the GcvA protein.

Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding.

The GcvA protein is required for both glycine-mediated activation and purine-mediated repression of the gcvTHP operon. Random and site-directed PCR mutagenesis was used to create nucleotide changes in gcvA to identify residues of the protein involved in activation, repression, and DNA binding. Single amino acid substitutions at L30 and F31 cause a defect in activation of a gcvT-lacZ fusion but have no effect on repression or DNA binding. Single amino acid substitutions at V32 and S38 cause the loss of binding of GcvA to DNA. A deletion of the carboxy-terminal 14 amino acids of GcvA results in the loss of purine-mediated repression and, consequently, a constitutive activation of a gcvT-lacZ fusion. The results of this study partially define regions of GcvA involved in activation, repression, and DNA binding and demonstrate that these functions of GcvA are genetically separable.

Promoter characterization and constitutive expression of the Escherichia coli gcvR gene.

The Escherichia coli glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (gcv). In this study, the gcvR translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the gcvR promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a gcvR-lacZ fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of gcv, respectively. In addition, it was shown that gcvR-lacZ expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream bcp gene overlaps the gcvR stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of gcvR had no effect on the expression of a downstream bcp-lacZ fusion, and primer extension analysis of the bcp promoter region demonstrated that bcp has its own promoter within the gcvR coding sequence. These results show that gcvR and bcp do not form an operon. Furthermore, the deletion of bcp from the chromosome had no effect on gcv-lacZ expression.

The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli.

A mutation in the rpoA gene (which encodes the alpha subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a lambda metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the -35 region of the metE promoter that interacts with the alpha subunit. Our data indicate that the glutamic acid at position 261 in the alpha subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.

MetR-mediated repression of the glyA gene in Escherichia coli.

Inactivation of either of the two MetR binding sites centered at bp -143 and 121 relative to the +1 transcription start site results in reduced glyA-lacZ expression in a wild-type strain below the level seen in a metR mutant. This reduced expression is dependent on the side of the DNA helix MetR binds relative to the RNA polymerase binding site. Thus, a single MetR dimer bound to the DNA may play a physiological role in maintaining appropriate glyA gene expression, functioning as a repressor under low MetR conditions.

RNA polymerase, PurR and MetR interactions at the glyA promoter of Escherichia coli.

In Escherichia coli, the MetR and PurR proteins positively and negatively regulate glyA gene expression, respectively. A DNase I footprint analysis showed that both proteins bind independently to the glyA control region. The PurR protein blocks RNA polymerase (RNAP) from binding to the glyA promoter. The presence of hypoxanthine, the co-repressor of PurR, increases the ability of PurR to prevent RNAP binding, providing a model for repression of the glyA gene by PurR. In contrast, MetR alters the RNAP footprint pattern of the glyA control region. In addition, the MetR footprint is increased in the presence of RNAP, suggesting that the two proteins might interact.

Cooperative MetR binding in the Escherichia coli glyA control region.

We determined the relative binding affinity of the MetR protein for wild-type and mutant MetR binding sites 1 and 2 in the Escherichia coli glyA control region. The results show that MetR binding site 1 has a higher affinity for the MetR protein than binding site 2. In addition, the results suggest that binding of MetR to the glyA promoter is cooperative. Mutations that decrease the ability of MetR to bind to either site 1 or site 2 have no significant effect on MetR's ability to bend DNA.

Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium.

Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens. An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice. The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis. S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid. Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine. Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

Propofol compatibility with other intravenous drug products: two new methods of evaluating IV emulsion compatibility.

OBJECTIVE: To determine the physical compatibility of propofol injection with 77 other drug products using two methods not previously reported. DESIGN: Each of 77 drug products were mixed with (1) an equal volume of propofol injection, (2) an equal volume of propofol injection with methylene blue added, and (3) an equal volume of the separated aqueous phase of propofol injection. The mixtures were observed for physical changes, and the turbidity of the aqueous phase mixtures was measured by nephelometry. SETTING: A university-affiliated urban teaching hospital. MAIN OUTCOME MEASURES: Observed physical changes or changes in turbidity. RESULT: Of the 77 drugs tested, 69 showed immediate evidence of physical change, and the other 8 were found incompatible within 72 hours. CONCLUSIONS: Practitioners should not mix the immediately incompatible products with propofol by administering them in the same intravenous line. Caution should be used in simultaneous administration of the other drugs. The new methods were useful in detecting incompatibilities in emulsions.

Escherichia coli cis- and trans-acting mutations that increase glyA gene expression.

We used an Escherichia coli strain blocked in serine biosynthesis and carrying a partial glyA deletion to isolate strains with altered regulation of the glyA gene. The glyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increased glyA expression were isolated on glycine supplemented plates. One class of mutations increased glyA expression 10-fold by directly altering the -35 consensus sequence of the glyA promoter. The two other classes increased glyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of the metE gene of the folate branch of the methionine pathway, but not metA in the nonfolate branch of the methionine pathway, or the gcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on the E. coli chromosome.

DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli.

The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA. Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions. DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from -28 to +20. Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions -271 to -242, a 28-bp region extending from -242 to -214, and a 35-bp region covering positions -69 to -34 relative to the transcription initiation site. PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv.

Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression.

The Escherichia coli glycine cleavage enzyme system catalyzes the cleavage of glycine, generating CO2, NH3, and a one-carbon unit. Expression of the operon encoding this enzyme system (gcv) is induced in the presence of glycine and repressed in the presence of purines. In this study, a mutant with high-level constitutive expression of a gcvT-lacZ gene fusion was isolated. The mutation in this strain was designated gcvR1 and was mapped to min 53.3 on the E. coli chromosome. A single-copy plasmid carrying the wild-type gcvR gene complemented the mutation, restoring normal regulation of a gcvT-lacZ fusion, while a multicopy plasmid carrying gcvR led to superrepression under all growth conditions. Negative regulation of a gcvT-lacZ fusion by GcvR was shown to require GcvA, a LysR family protein known to both activate gcv in the presence of glycine and repress gcv in the presence of purines. Models explaining how GcvR and GcvA might interact to regulate gcv expression are proposed.

Characterization of the MetR binding sites for the glyA gene of Escherichia coli.

Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein. Gel shift assays and DNase I protection assays verified that both sites bind MetR. Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region. The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees. MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression. Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation.

Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium.

Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator. It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides -48 to -71 relative to the metE transcription initiation site (designated as site 1). In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides -24 to -47 relative to the metE transcription initiation site (designated as site 2). Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion. Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression. DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2. A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.

A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli.

The DNA-binding protein MetR belongs to the LysR family of transcriptional activators and is required for expression of the metE and metH promoters in Escherichia coli. However, it is not known if this activation is mediated by a direct interaction of MetR with RNA polymerase. In a search for RNA polymerase mutants defective in MetR-mediated activation of the metE gene, we isolated a mutation in the alpha subunit of RNA polymerase that decreases metE expression independently of the MetR protein. The mutation does not affect expression from the metH promoter, suggesting that the alpha subunit of RNA polymerase interacts differently at these two promoters. The mutation was mapped to codon 261 of the rpoA gene, resulting in a change from a glutamic acid residue to a lysine residue. Growth of the mutant is severely impaired in minimal medium even when supplemented with methionine and related amino acids, indicating a pleiotropic effect on gene expression. This rpoA mutation may identify either a site of contact with an as yet unidentified activator protein for metE expression or a site of involvement by the alpha subunit in sequence-specific recognition of the metE promoter.

The Escherichia coli glycine transport system and its role in the regulation of the glycine cleavage enzyme system.

An Escherichia coli K12 mutant defective in both serine biosynthesis (serA) and glycine transport (cycA) was found to exhibit a glycine cleavage negative (GCV-) phenotype, i.e. was unable to use glycine as a serine source. While [2-14C]glycine uptake and induction of a lambda gcvT::lacZ fusion were greatly reduced in a cycA mutant compared to the wild-type, both strains exhibited parallel increases in uptake and induction with increasing exogenous glycine concentrations. A plasmid carrying the wild-type cyc region complemented the GCV- phenotype and restored both glycine uptake and glycine-inducible gcvT::lacZ expression. Wild-type and cycA strains grown in the presence of either a glycine-containing tripeptide or threonine, which can be degraded internally into glycine, exhibited similar induction of the gcvT::lacZ fusion. However, when a gcv mutation, which causes glycine to accumulate within the cell, was introduced into the cycA strain, there was increased induction of the gcvT::lacZ fusion, but induction was less than that observed in a gcv cycA+ strain. It is proposed that cyc serves primarily in the regulation of gcv by transporting glycine into the cell, which endogenously induces gcv expression. However, the possibility of some form of exogenous regulation of gcv, mediated by the cyc-encoded glycine transport system, exists.