Biomedical subjects

C A Gross

Publications and source records attributed to C A Gross.

At least 19 recordsLinked to original sources

PubMed1997

The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways.

The activity of the alternate sigma-factor sigmaE of Escherichia coli is induced by several stressors that lead to the extracytoplasmic accumulation of misfolded or unfolded protein. The sigmaE regulon contains several genes, including that encoding the periplasmic protease DegP, whose products are thought to be required for maintaining the integrity of the cell envelope because cells lacking sigmaE are sensitive to elevated temperature and hydrophobic agents. Selection of multicopy suppressors of the temperature-sensitive phenotype of cells lacking sigmaE revealed that overexpression of the lipoprotein NlpE restored high temperature growth to these cells. Overexpression of NlpE has been shown previously to induce DegP synthesis by activating the Cpx two-component signal transduction pathway, and suppression of the temperature-sensitive phenotype by NlpE was found to be dependent on the Cpx proteins. In addition, a constitutively active form of the CpxA sensor/kinase also fully suppressed the temperature-sensitive defect of cells lacking sigmaE. DegP was found to be necessary, but not sufficient, for suppression. Activation of the Cpx pathway has also been shown to alleviate the toxicity of several LamB mutant proteins. Together, these results reveal the existence of two partially overlapping regulatory systems involved in the response to extracytoplasmic stress in E. coli.

Bacterial Outer Membrane Proteins↗

PubMed1997

The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE.

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, sigma(E). sigma(E) activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that sigma(E) activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25-fold induction of sigma(E) activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits sigma(E)-directed transcription in vitro, indicating that RseA acts as an anti-sigma factor. Deletion of rseB leads to a slight induction of sigma(E), indicating that RseB is also a negative regulator of sigma(E). RseB is a periplasmic protein and was found to co-purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on sigma(E) through RseA. Deletion of rseC, in contrast, has no effect on sigma(E) activity under steady-state conditions. Under induction conditions, strains lacking RseB and/or C show wild-type induction of sigma(E) activity, indicating either the presence of multiple pathways regulating sigma(E) activity, or the ability of RseA alone to both sense and transmit information to sigma(E).

Bacterial Outer Membrane Proteins↗

PubMed1997

SigmaE is an essential sigma factor in Escherichia coli.

SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.

Escherichia coli↗

PubMed1996

SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins.

Little is known about either the process of periplasmic protein folding or how information concerning the folding state in this compartment is communicated. We present evidence that SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, is involved in the maturation and assembly of LamB. LamB is a trimeric outer membrane porin for maltodextrins as well as the bacteriophage lambda receptor in Escherichia coli. We demonstrate that SurA is involved in the conversion of unfolded monomers into a newly identified intermediate in LamB assembly, which behaves as a folded monomer. The absence of SurA blocks the assembly pathway and leads to accumulation of species prior to the folded monomer. These species also accumulate when the stress sigma factor sigmaE is induced by LamB overexpression. We suggest that accumulation of species prior to the generation of folded monomer is a stress signal sensed by sigmaE.

Amino Acid Isomerases↗

PubMed1996

Sigma domain structure: one down, one to go.

The recent publication of the 2.6 A crystal structure of a portion of sigma70 provides insight into the role of sigma during transcription initiation. This high resolution picture unveils novel questions.

Bacterial Proteins↗

PubMed1996

The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing.

The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.

Base Sequence↗

PubMed1996

Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex.

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.

Amino Acid Sequence↗

PubMed1996

Identifying interacting regions in the beta subunit of Escherichia coli RNA polymerase.

Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves. As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues. We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E). With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit. Allele specific suppression is a genetic criterion for protein-protein interaction. Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation. Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide. We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme.

Alleles↗

PubMed1996

A structure/function analysis of Escherichia coli RNA polymerase.

Control of RNA polymerase is a common means of regulating gene expression. A detailed picture of both the structure and how the structural details of RNA polymerase encode function is a key to understanding the molecular strategies used to regulate RNA polymerase. We review here data which ascribes functions to some regions of the primary sequence of the subunits (alpha, beta beta' sigma) which make up E. coli RNA polymerase. We review both genetic and biochemical data which place regions of the primary sequence that are distant from one another in close proximity in the tertiary structure. Finally we discuss the implications of these findings on the quaternary structure of RNA polymerase.

DNA-Directed RNA Polymerases↗

PubMed1996

Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli.

We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways. Our previous finding that SecB-deficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway. Using depletion studies, we now demonstrate a requirement for GrpE in this pathway. In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and beta-lactamase), they are not the primary chaperones in this process.

Alkaline Phosphatase↗

PubMed1996

Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli.

The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.

Amino Acid Sequence↗

PubMed1995

Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes.

DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.

Adenosine Triphosphatases↗

PubMed1995

rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli.

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

Amino Acid Sequence↗

PubMed1995

Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica.

We previously reported that a region of the Escherichia coli chromosome at 18 min increased E sigma E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618-2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in E sigma E activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, E sigma E activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, E sigma E activity was dependent on expression of OmpX in certain strain backgrounds.

Amino Acid Sequence↗

PubMed1995

Clinical safety of gadopentetate dimeglumine.

PURPOSE: To quantify the rate of adverse reactions to gadopentetate dimeglumine. MATERIALS AND METHODS: Magnetic resonance (MR) imaging was performed in 15,496 patients in April-September 1992. Data were collected before and after intravenous administration of 0.1 mmol/kg gadopentetate dimeglumine. RESULTS: Adverse reactions occurred in 2.4% (n = 372) of patients. Symptoms abated the same or next day in 94.1% (n = 350). Whereas onset occurred within 30 minutes after injection in 49.7% (n = 185), onset occurred more than 1 hour after injection in 44.9% (n = 167). Two serious adverse reactions occurred and were attributed to underlying disease. The rate of adverse reaction was 3.7% in patients with a history of asthma (31 of 831 patients) or allergy (144 of 3,860 patients). Patients with previous reactions to an MR imaging or iodinated contrast agent had an adverse-reaction rate in this study of 21.3% (16 of 75) and 6.3% (54 of 857), respectively. The rate of adverse reaction was 2.2% when gadopentetate dimeglumine was administered slowly and 2.9% when it was administered rapidly. CONCLUSION: Findings confirm the safety of gadopentetate dimeglumine.

Asthma↗

PubMed1993

Four contiguous amino acids define the target for streptolydigin resistance in the beta subunit of Escherichia coli RNA polymerase.

Streptolydigin (stl), a bacteriostatic inhibitor of transcription elongation, interacts with the beta subunit of Escherichia coli RNA polymerase. We have defined the target for stl resistance using chemical mutagenesis and mutagenic polymerase chain reaction. Mutations resulting in stl resistance are confined to a small cluster of contiguous amino acids, amino acids 543 to 546. These stlr mutants differ from one another in their levels of resistance to stl in vivo and in vitro. We have analyzed two of the mutants, A543V and F545S, for their effects on elongation and termination in vivo and in vitro. Neither affected termination at rho-dependent or rho-independent terminators. These mutants were indistinguishable from wild type in a T7 in vitro elongation assay. F545S, however, did exhibit slower elongation kinetics in a lambda tR1 pausing assay. We conclude that mutations in the stlr region can influence transcription elongation, but that these amino acids are not directly involved in catalysis.

Amino Acid Sequence↗

PubMed1993

Genetic evidence for the interaction between cluster I and cluster III rifampicin resistant mutations.

Rifampicin-resistant (Rifr) mutations of Escherichia coli map to the central portion of the rpoB gene, which encodes the beta subunit of RNA polymerase. These mutations are located in three distinct clusters, designated I, II and III. Three intragenic suppressors of the cluster III Rifr mutation, rpoB3406(RH687), restore the ability of the mutant strain to grow at low and high temperatures and map to a single locus in cluster I. These suppressors are identical to two previously characterized Rifr alleles, rpoB3401(RC529) and rpoB3402(RS529). None of the other 14 previously identified Rifr mutations that we have characterized confers this phenotype. We suggest that this allele-specific suppression results from interaction between Cluster I and Cluster III of the beta subunit.

Alleles↗

PubMed1993

Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase. Genetic studies have indicated that sigma itself interacts with DNA at the -10 and -35 promoter consensus sequences. Binding of Escherichia coli sigma 70 to DNA in vitro, however, can only be observed for truncated polypeptides lacking the amino-terminal amino acids. We have investigated the role of the amino terminus of E. coli sigma 70 in controlling DNA-binding ability. Deletion analysis indicates that amino acids within amino-terminal region 1.1 of sigma 70 inhibit DNA binding by the carboxy-terminal DNA-binding domains. Furthermore, inhibition of binding by the amino-terminal inhibitory domain of sigma 70 can be observed in trans. Likewise, the amino-terminal extensions of two alternative sigma-factors, E. coli sigma 32 and Bacillus subtilis sigma K, negatively affect the DNA binding activity of their carboxy-terminal domains. We propose that initiation of transcription is subject to modulation as a result of the composition and/or structure of the amino terminus of the sigma-subunit and that the sigma family of proteins belong to a larger class of intramolecularly regulated transcriptional effectors.

Amino Acids↗