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Biomedical subjects

S Gottesman

Publications and source records attributed to S Gottesman.

At least 19 recordsLinked to original sources
PubMed1992

Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12.

Deletions which removed rfa genes involved in lipopolysaccharide (LPS) core synthesis were constructed in vitro and inserted into the chromosome by linear transformation. The deletion delta rfa1, which removed rfaGPBI, resulted in a truncated LPS core containing two heptose residues but no hexose and a deep rought phenotype including decreased expression of major outer membrane proteins, hypersensitivity to novobiocin, and resistance to phage U3. In addition, delta rfa1 resulted in the loss of flagella and pili and a mucoid colony morphology. Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion showed that the mucoid phenotype was due to rcsC-dependent induction of colanic acid capsular polysaccharide synthesis. Complementation of delta rfa1 with rfaG+ DNA fragments resulted in a larger core and restored the synthesis of flagella and pili but did not reverse the deep rough phenotype or the induction of cps-lacZ, while complementation with a fragment carrying only rfaP+ reversed the deep rough phenotype but not the loss of flagella and pili. A longer deletion which removed rfaQGPBIJ was also constructed, and complementation studies with this deletion showed that the product of rfaQ was not required for the functions of rfaG and rfaP. Thus, the function of rfaQ remains unknown. Tandem mass spectrometric analysis of LPS core oligosaccharides from complemented delta rfa1 strains indicated that rfaP+ was necessary for the addition of either phosphoryl (P) or pyrophosphorylethanolamine (PPEA) substituents to the heptose I residue, as well as for the partial branch substitution of heptose II by heptose III. The substitution of heptose II is independent of the type of P substituent present on heptose I, and this results in four different core structures. A model is presented which relates the deep rough phenotype to the loss of heptose-linked P and PPEA.

Carbohydrate Sequence↗
PubMed1992

Regulation by proteolysis: energy-dependent proteases and their targets.

A number of critical regulatory proteins in both prokaryotic and eukaryotic cells are subject to rapid, energy-dependent proteolysis. Rapid degradation combined with control over biosynthesis provides a mechanism by which the availability of a protein can be limited both temporally and spatially. Highly unstable regulatory proteins are involved in numerous biological functions, particularly at the commitment steps in developmental pathways and in emergency responses. The proteases involved in energy-dependent proteolysis are large proteins with the ability to use ATP to scan for appropriate targets and degrade complete proteins in a processive manner. These cytoplasmic proteases are also able to degrade many abnormal proteins in the cell.

ATP-Dependent Proteases↗
PubMed1991

A mechanism for maintaining an up-to-date GenBank database via Usenet.

In this paper, we describe an automated system for distributing updates to the GenBank nucleic acid sequence database, using the Usenet news system as the underlying transport mechanism. Our system allows new loci to be distributed as soon as the sequences are available, over existing networks, using existing Usenet software and infrastructure currently available on a wide range of computer systems.

Amino Acid Sequence↗
PubMed1991

A simple approach for the distribution of computationally intense tasks in an heterogeneous environment: distribution of the MDPP image-processing package.

A method has been developed to link the display ability of a high-resolution graphics workstation with the computational power of a local mainframe or a remote supercomputer via an electronic data network. The method allows this link to be established in a manner largely transparent to the user. The application of the method is illustrated by our successful distribution of the computationally intensive portions of an imaging program (MDPP) from a small VAX workstation to a VAX mainframe and Cray Y-MP8/832 using a simple message-passing technique. This technique can be applied to almost any configuration of networked machines.

Algorithms↗
PubMed1991

Regulation of capsular polysaccharide synthesis in Escherichia coli K12.

Synthesis of the capsular polysaccharide colanic acid in Escherichia coli K12 is regulated by a complex network of regulatory proteins. This regulation is expressed at the level of transcription of the cps (capsular polysaccharide synthesis) genes. Two positive regulators, RcsA and RcsB, are necessary for maximal capsule expression. The availability of RcsA is normally limited because the RcsA protein is rapidly degraded by the Lon ATP-dependent protease. Therefore Lon acts, indirectly, as a negative regulator of capsule synthesis. The sequence predicted for RcsB suggests that it is the effector component of a two-component system; a protein with homology to sensors, RcsC, also plays a role in capsule regulation. We propose a model for capsule synthesis in which RcsA interacts with RcsB to stimulate transcription of the cps genes. The mechanism of regulation of colanic acid synthesis in E. coli may apply to other capsules in a variety of Gram-negative bacteria.

ATP-Dependent Proteases↗
PubMed1991

RcsA, an unstable positive regulator of capsular polysaccharide synthesis.

RcsA is an unstable positive regulator required for the synthesis of colanic acid capsular polysaccharide in Escherichia coli. Degradation of the RcsA protein in vivo depends on the ATP-dependent Lon protease. DNA sequence analysis of the rcsA gene reveals a single open reading frame for a 23,500-Da highly basic protein (pI = 9.9), consistent with the observed size of the purified subunit of RcsA. The DNA and protein sequences are highly homologous to the rcsA gene and protein from Klebsiella pneumoniae and other species. The carboxy-terminal region of RcsA contains a possible helix-turn-helix DNA-binding motif that resembles sequences found at the carboxy terminus of RcsB, another positive regulator of capsule synthesis, and in several other transcriptional regulators including members of the LuxR family. rcsA62, a mutation in rcsA that leads to increased capsule synthesis, encodes a protein designated RcsA*, which differs from wild-type RcsA only in the replacement of Met-145 by valine. The RcsA* protein is subject to Lon-dependent degradation. The stability of wild-type RcsA in vivo is increased by multicopy RcsB. Conversely, RcsA is degraded more rapidly in rcsB mutant hosts than in wild-type hosts. These results suggest that RcsA and RcsB interact in vivo and are consistent with genetic experiments that indicate an interaction between RcsA and RcsB. Based on these experiments, we propose a model for capsule regulation in which RcsA interacts directly with RcsB to promote transcription of the genes for capsule synthesis.

Amino Acid Sequence↗
PubMed1990

Sequence and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli.

The ATP-dependent Clp protease of Escherichia coli contains two dissimilar components: the Clp A regulatory polypeptide, with two ATP binding sites and intrinsic ATPase activity, and the Clp P subunit, which contains the proteolytic active site. The DNA sequence of the clpP gene predicts a protein of 207 amino acids (Mr 21,679), which is in close agreement with the size determined by sodium dodecyl sulfate-gel electrophoresis of purified Clp P. Clp P has a native Mr of approximately 240,000, and electron micrographs of the protein show superimposed disk-like structures with a central cavity, similar in appearance to purified proteasomes from eukaryotic cells. Clp P is synthesized with a 14-amino acid leader which is rapidly cleaved in vivo to yield the 193-amino acid protein which has activity in vitro. The clpP gene is at 10 min on the E. coli map, close to that for the ATP-dependent Lon protease of E. coli and far from the gene for clpA. Primer extension experiments indicate that transcription initiates immediately upstream of the coding region for Clp P, with a major transcription start at 120 bases in front of the start of translation. Insertion mutations in clpP have been isolated and transferred to the chromosome; strains devoid of Clp P are viable in the presence or absence of Lon protease. Mutations in clpP stabilize the same Clp A-beta-galactosidase fusion protein specifically stabilized by clpA mutations, providing the first genetic evidence that Clp A and Clp P act together in vivo.

ATP-Dependent Proteases↗
PubMed1990

Clp P represents a unique family of serine proteases.

The amino acid sequence of Clp P, the proteolytic subunit of the ATP-dependent Clp protease of Escherichia coli, closely resembles a protein encoded by chloroplast DNA, which is well conserved between chloroplasts of different plant species. The homology extends over almost the full length of the sequences of both proteins and consists of approximately 46% identical and approximately 70% similar amino acids. Antibodies against E. coli Clp P cross-reacted with proteins with Mr of 20,000-30,000 in bacteria, lower eukaryotes, plants, and animal cells. Since the regulatory subunit of Clp protease, Clp A, also has a homolog in plants, as well as in other bacteria and in lower eukaryotes, it is likely that ATP-dependent proteolysis in chloroplasts is catalyzed in part by a Clp-like protease and that both components of Clp-like proteases are widespread in living cells. We have identified Ser-111 as the active site serine in E. coli Clp P modified by diisopropyl fluorophosphate. Mutational alteration of Ser-111 or His-136 eliminates proteolytic activity of Clp P. Both residues are found in highly conserved regions of the protein. The sequences around the active site residues suggest that Clp P represents a unique class of serine protease. Amino-terminal processing of cloned Clp P mutated at either Ser-111 or His-136 occurs efficiently when wild-type clpP is present in the chromosome but is blocked in clpP- hosts. Processing of Clp P appears, therefore, to involve an intermolecular autocatalytic cleavage reaction. Since processing of Clp P occurs in clpA- cells, the autoprocessing activity of Clp P is independent of Clp A.

ATP-Dependent Proteases↗
PubMed1990

The ATP-dependent Clp protease of Escherichia coli. Sequence of clpA and identification of a Clp-specific substrate.

The clpA gene, which codes for the ATP-binding subunit of the ATP-dependent Clp protease of Escherichia coli, has been sequenced. The coding region contains a single open reading frame for a protein of 758 amino acids; within the amino acid sequence are two consensus sequences for ATP-binding sites. The sequence of ClpA does not resemble that of other previously described ATPases or Lon, the other sequenced ATP-dependent protease of E. coli, except in the ATP-binding site consensus region. The clpA gene is expressed as a monocistronic message. Primer extension experiments define a major start point of transcription at -183 relative to the start of translation. A rho-independent terminator is located 23 bases beyond the end of the coding region. The ClpA protein is degraded in vivo in a Clp-dependent fashion (t1/2 approximately 60 min). A fusion protein containing the first 40 amino acids of ClpA fused in frame to beta-galactosidase is degraded very rapidly in a clpA+ host (t1/2 approximately 3 min) but not in a clpA- host. This fusion protein is the first Clp-specific substrate described.

ATP-Dependent Proteases↗
PubMed1990

Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes.

Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coli, Clp. All members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding. Within these regions there is at least 85% similarity between the most distant members of the family. The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells.

ATP-Dependent Proteases↗
PubMed1990

On the bacterial cell cycle: Escherichia coli mutants with altered ploidy.

We describe a scheme for isolation of new classes of mutants in the cell cycle of Escherichia coli. The mutants were selected as resistant to camphor vapors, which results in increased ploidy, and were subsequently screened for an increase in cell density and an increase in the gene dosage of the lac operon. Our mutations are located at four different places in the chromosome; we have named these loci mbr (moth ball resistant). mbrA maps to 68 min on the E. coli chromosome, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD to 90 min. mbrD mutations may be alleles of rpoB (a subunit of RNA polymerase). In addition to the selected or screened phenotypes, most of the mutants fail to grow on rich media or at high temperatures. We have examined the nine mutants under nonpermissive conditions, using several techniques to determine the cause of death. We have also coupled our mutations with lesions in dnaA, which is required for cell-cycle-specific DNA replication, and rnh (the gene for RNase H), which is required for specificity in the DNA initiation reaction, and determined the effects of the double and triple mutants under permissive and nonpermissive conditions. These tests have shown that bacteria mutated at mbrA do not tolerate a null mutation in rnh, indicating that they are dependent on DNA replication initiating at oriC. In contrast, mutations at mbrB, mbrC, and mbrD exhibit their phenotypes independent of oriC initiation of DNA replication, suggesting that the mutations affect factors that influence the DNA/cell ratio regardless of the origin of DNA replication. Based on our results, the mbr mutations appear to have defects in cell-cycle timing and/or defects in chromosomal partitioning.

Camphor↗
PubMed1990

RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli.

Colanic acid capsule synthesis in Escherichia coli K-12 is regulated by RcsB and RcsC. The amino acid sequences of these two proteins, deduced from the nucleotide sequence reported here, demonstrate their homology to environmentally responsive two-component regulators that have been reported in both gram-positive and gram-negative bacteria. In our model, RcsC acts as the sensor and RcsB acts as the receiver or effector to stimulate capsule synthesis from cps genes. In addition, RcsC shows limited homology to the other effectors in its C terminus. Fusions of rcsC to phoA that resulted in PhoA+ strains demonstrated that RcsC is a transmembrane protein with a periplasmic N-terminal domain and cytoplasmic C-terminal domain. Additional control of this regulatory network is provided by the dependence on the alternate sigma factor, RpoN, for the synthesis of RcsB. The rcsB and rcsC genes, which are oriented convergently with their stop codons 196 base pairs apart, are separated by a long direct repeat including two repetitive extragenic palindromic sequences.

Amino Acid Sequence↗
PubMed1989

Autoradiographic localization of [3H]dextromethorphan in guinea pig brain: allosteric enhancement by ropizine.

Dextromethorphan (DM) is an antitussive with anticonvulsant activity that binds to high- and low-affinity sites in guinea pig brain homogenates. We examined the autoradiographic localization of [3H]DM using the anticonvulsant ropizine, an allosteric modifier that decreases the dissociation rate of [3H]DM. Competition studies demonstrated that the binding to brain sections was identical to that of brain homogenates [Craviso and Musacchio: Mol Pharmacol 23:629-640, 1983b]. Computer-assisted quantitative analysis of the autoradiographic images demonstrated that [3H]DM binds to discrete structures throughout the brain, but with higher density in the midbrain, pons, and medulla oblongata. The most intense labeling was observed in the rhabdoid, dorsal raphe, median raphe, caudal linear raphe nuclei, and cranial motor nerve nuclei. The central gray showed moderate to high-density labeling throughout its entire rostro-caudal extent, with very high binding in the dorsal tegmental nucleus and the locus coeruleus. Moderate and high binding was also seen in several hypothalamic structures. Distinct bands of moderate binding were seen in the pyramidal cell layer of the piriform cortex, the retrosplenial cortex, the granular cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the Purkinje cell layer of the cerebellum. The striking similarity between the binding distribution of [3H]DM and sigma ligands, plus competition studies in brain homogenate, support the hypothesis that DM and sigma ligands share a common high-affinity binding site [Musacchio et al: Mol Pharmacol 35:1-5, 1989]. The distribution of [3H]DM binding provides possible anatomical substrates for both the antitussive and anticonvulsant actions of DM.

Allosteric Regulation↗
PubMed1989

Alp, a suppressor of lon protease mutants in Escherichia coli.

Escherichia coli lon mutants lack a major ATP-dependent protease, are sensitive to UV light and methylmethane sulfonate (MMS), and overproduce capsular polysaccharide. Evidence is presented that an activity (Alp), cloned on a multicopy plasmid, can suppress the phenotypes of lon mutants. The sensitivity to UV and MMS is a reflection of the stabilization of the cell division inhibitor SulA, while the capsule overproduction arises through the stabilization of a transcriptional activator of capsule biosynthetic genes, RcsA. Multicopy alp (pAlp) suppressed capsule formation in delta lon cells, and delta lon cells containing the pAlp plasmid were resistant to MMS treatment. The MMS resistance of delta lon pAlp+ cells correlates with an increase in the degradation of SulA to that found in lon+ cells. Lon-directed degradation of SulA was energy dependent, as was the increase in degradation of SulA in delta lon pAlp+ cells. alp maps close to pheA, at 57 min on the E. coli chromosome. Although pAlp can substitute for Lon, cells lacking alp activity did not have the phenotype on a lon mutant. This study demonstrates that at least one activity, when overproduced in the cell, can substitute for Lon protease.

ATP-Dependent Proteases↗