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S Gottesman

Publications and source records attributed to S Gottesman.

At least 73 records · Page 4Linked to original sources
PubMed1987

A multiple-component, ATP-dependent protease from Escherichia coli.

A new ATP-dependent, casein-degrading proteolytic complex has been identified and partially purified from Escherichia coli. The proteolytic complex can be isolated from wild-type cells as well as from mutants in which the gene for the ATP-dependent Lon protease is deleted. The complex consists of at least two components (components I and II) that can be separated from each other (and from wild-type Lon protease) by phosphocellulose chromatography. Neither component has casein-degrading activity when added separately to assay solutions with or without ATP. Both components must be present simultaneously for casein degradation to occur. Of the nucleotides tested, only ATP activates the proteolytic complex, and the ATP must be present continuously for degradation to occur. Component II copurifies with an ATPase activity and binds to a Type 4 ATP affinity column. ATP protects component II from heat inactivation, suggesting that component II interacts with ATP. Proteolysis was not inhibited by any serine protease inhibitors but was inhibited by reagents such as the organomercurial Neohydrin and N-ethylmaleimide, which react with sulfhydryl groups. Our data provide convincing evidence that E. coli possesses a previously undescribed proteolytic system composed of at least two complementary components and absolutely dependent on ATP.

ATP-Dependent Proteases↗
PubMed1987

Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function.

A primary determinant of pathogenicity in Erwinia stewartii is the production of extracellular polysaccharide (EPS). A single mutation can abolish both EPS synthesis and pathogenicity; both properties are restored by a single cosmid clone. Subcloning and insertion analysis have defined a single positive regulatory function which shares a number of similarities with the rcsA function of Escherichia coli K-12, a positive regulator for capsular polysaccharide synthesis. In E. stewartii, the gene promotes the transcription of at least two operons (cps) involved in EPS synthesis; we have previously demonstrated a similar function for rcsA in E. coli. Both genes code for proteins of 25 to 27 kilodaltons; both proteins are unstable in E. coli. The E. stewartii RcsA protein was stabilized in E. coli lon mutants, as the RcsA product from E. coli is. The E. stewartii function complemented E. coli rcsA mutants, and the E. coli RcsA function increased cps expression and restored virulence in E. stewartii mutants. Therefore, these two gram-negative organisms share a similar component of their regulatory circuitry for the control of capsular polysaccharide synthesis.

Bacterial Proteins↗
PubMed1987

Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis.

lon mutants of Escherichia coli K-12 are defective in an ATP-dependent protease, are UV sensitive, and overproduce the capsular polysaccharide colanic acid. Six structural genes needed for capsular polysaccharide synthesis (cps) are transcriptionally regulated by lon as well as by three other regulatory genes, rcsA, -B, and -C (S. Gottesman, P. Trisler, and A. S. Torres-Cabassa, J. Bacteriol. 162:1111-1119, 1985). We have cloned rcsA, the gene for a positive regulator of capsule synthesis, onto multicopy plasmids and defined the gene by both insertions and deletions. The product of rcsA has been identified as an unstable protein of 27 kilodaltons. RcsA has a half-life of 5 min in lon+ cells and one of 20 min in lon cells. The availability of RcsA is the limiting factor for capsule synthesis; doubling the gene dosage of rcsA+ significantly increases expression of cps genes. Our results are consistent with a model in which the presence of a lon mutation increases the synthesis of capsular polysaccharide via stabilization of RcsA.

ATP-Dependent Proteases↗
PubMed1986

Failure to confirm the existence of short-latency, short-loop feedback regulation (autoregulation) of growth hormone secretion in the human.

That growth hormone (GH) regulates its own secretion by negative feedback both directly and indirectly via somatomedins has been well-documented in the rat and assumed, on the basis of limited studies, to be true for man as well. Prior to proceeding with studies designed to investigate the nature of this feedback in various endocrine states, we sought first to confirm the existence of direct, short-latency GH feedback in normal individuals. Primed, continuous rate infusions of human GH in normal volunteers achieved a range of steady state GH levels. After 1 h of GH infusion, arginine HCl (500 mg/kg) was infused over 30 min and the GH response assessed. Seven of 8 subjects achieved steady state GH levels in the 8-21 ng/ml range with an infusion rate of 0.0045 U/min following a 0.277 U bolus. After arginine, there was a significant increment of GH levels (range 11.6-69.5 ng/ml) in all 7 subjects. With a higher infusion rate of 0.009 U/min following a 0.54 U bolus, 1 of 2 subjects reached a steady state of 31.0 ng/ml and no response to arginine was demonstrable. Two subjects reached steady state levels of 45.0 and 58 ng/ml during a 0.018 U/min infusion after a 1.08 U bolus and had increments of 26.2 and 25.9 ng/ml following arginine. In 3 subjects achieving levels of 64.5, 107.0 and 132.0 ng/ml, there were increments of 55.0, 61.7 and 13.0 ng/ml during infusions of 0.036 U/min following boluses of 2.16 U. However, only in the first of these 3 was a true steady state achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1985

Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes.

The synthesis of the Escherichia coli capsular polysaccharide varies with growth medium, temperature of growth, and genetic background. lac fusions to genes necessary for capsule synthesis (cps) demonstrated that these genes are regulated negatively in vivo by the lon gene product. We have now isolated, characterized, and mapped mutations in three new regulatory genes (rcs, for regulator of capsule synthesis) that control expression of these same fusions. rcsA and rcsB are positive regulators of capsule synthesis. rcsA is located at min 43 on the E. coli map, whereas rcsB lies at 47 min. rcsC, a negative regulator of capsule synthesis, is located at min 47, close to rcsB. All three regulatory mutations are unlinked to either the structural genes cpsA-F or lon. Mutations in all three rcs genes are recessive to the wild type. We postulate that lon may regulate capsule synthesis indirectly, by regulating the availability of one of the positive regulators.

Chromosome Mapping↗
PubMed1985

Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.

Adenosine Triphosphate↗
PubMed1984

Cell-division control in Escherichia coli: specific induction of the SOS function SfiA protein is sufficient to block septation.

Blocks in DNA replication cause a rapid arrest of cell division in Escherichia coli. We have previously established that the function SfiA (SulA), induced under these conditions as part of the SOS response, is involved in this inhibition of division. To separate the effects of SfiA from those of other SOS functions, we have constructed a plac-sfiA operon fusion, permitting specific induction of SfiA protein by addition of the lac operon inducer isopropyl beta-D-thiogalactopyranoside (IPTG). In lon mutants, in which the unstable SfiA protein has a longer half-life, IPTG caused a rapid arrest of cell division. Under these conditions, there is no concomitant induction of the SOS response. IPTG also caused a rapid arrest of cell division in lon+ strains. These results demonstrate that induction of the SfiA protein is sufficient to cause inhibition of division. Mutations in the sfiB gene can suppress IPTG-induced SfiA-mediated inhibition of division. At higher SfiA concentrations, however, even sfiB mutants cease division; an additional mutation genetically inseparable from sfiB restores normal division. These observations reinforce the hypothesis that the SfiB protein, probably required for cell septation, is the target of action of the SfiA division inhibitor.

Bacterial Proteins↗
PubMed1984

lon transcriptional regulation of genes necessary for capsular polysaccharide synthesis in Escherichia coli K-12.

It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A. Markovitz, p. 415-462, in I. Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977). To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M. J. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains. These genes are located in a cluster close to udk at 45 min on the E. coli map; we have named these genes cpsA, B, C, D, and E. An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis. Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus. Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway.

DNA Restriction Enzymes↗
PubMed1983

Transcription of the sulA gene and repression by LexA.

The Escherichia coli sulA gene product is a highly unstable protein, whose synthesis in response to DNA damage is associated with an inhibition of septation. Genetic evidence as well as sequence information suggests that the sulA gene is part of the E. coli SOS system and is induced after DNA damage. We have constructed a plasmid carrying only the sulA gene; this plasmid is stable only when it contains an amber mutation in the sulA structural gene. Using fragments of this plasmid, we have carried out in vitro transcription experiments and demonstrated one major start site for RNA transcription. We have mapped this initiation point to an adenylate residue 30 nucleotides before the protein start. Purified LexA protein completely abolishes this transcription, in agreement with the prediction made from the genetic and sequence information previously available.

Bacterial Proteins↗
PubMed1983

lambda mutation in the Escherichia coli rho gene that inhibits the N protein activity of phage lambda.

Certain Escherichia coli rho mutations, exemplified by rho026, block the growth of phage lambda by interfering with phage gene expression. The phage gene N, whose product suppresses transcription termination, appears to be expressed normally in the mutants, and the functional stability of the N protein is not affected. Our data suggest that these rho mutations allow transcription to terminate despite the presence of N. Other E. coli mutants displaying a similar phenotype (Nus(-)) fail to propagate wild-type lambda but permit the growth of the lambda variant lambdanin5, which has undergone a deletion of the lambda terminator t(R2). The phenotype of the rho026 mutant differs: the growth of lambda is only marginally improved by the nin5 deletion. Interestingly, N activity at rho-independent terminators is not inhibited by the mutations, whereas its ability to suppress rho-dependent terminators is markedly reduced. The relevance of this specificity in terms of models of N action is discussed.

Bacteriophage lambda↗
PubMed1983

Protein degradation in Escherichia coli: the lon gene controls the stability of sulA protein.

Escherichia coli lon mutants are defective in the ATP-dependent proteolysis of abnormal proteins. The mutants are also sensitive to ultraviolet light (UV) in that septation is inhibited after exposure to UV. sulA mutations, isolated as suppressors of UV sensitivity unlinked to lon, do not affect proteolysis but allow septation to occur after DNA damage. We have confirmed the hypothesis that the product of the sulA gene is degraded by lon proteolysis. If sulA (the product of sulA) is a UV-inducible division inhibitor, as suggested by a variety of experiments, lon (the product of lon) may regulate cell division by regulating the half-life of sulA. We cloned the sulA gene in a bacteriophage lambda vector from a plasmid carrying the ompA region of E. coli. An 18-kilodalton polypeptide was identified as the product of the sulA gene. Pulse-chase labeling demonstrated that the half-life of the sulA protein is 1.2 min in lon+ cells and 19 min in lon- cells. This work demonstrates that lon proteolysis affects the stability of a native E. coli protein.

Bacteriophage lambda↗
PubMed1982

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination.

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.

Bacteriophage lambda↗
PubMed1981

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability.

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.

Bacterial Proteins↗