Larissa Brown

Eunice Kennedy Shriver National Institute of Child Health and Human Development

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Michael Cashel

Eunice Kennedy Shriver National Institute of Child Health and Human Development

Thomas Elliott

Medical Research Council

Jacob Humphrey

John Wiley & Sons (United States)

Linda Antas

Laura Routt

Sarah Bahauddin

Scott Ligocki

Felix Skowronek

Daniel R. Gentry

GlaxoSmithKline (United States)

Undine Mechold

Centre National de la Recherche Scientifique

Cooperative Institutions

National Institutes of Health

Jena University Hospital

West Virginia University

Eunice Kennedy Shriver National Institute of Child Health and Human Development

Pennsylvania State University

Western University

Health Sciences Centre

John Wiley & Sons (United States)

University of Vermont

University of Gdańsk

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DksA Affects ppGpp Induction of RpoS at a Translational Level

Journal of Bacteriology (2002)

Larissa Brown Daniel R. Gentry Thomas Elliott Michael Cashel

ABSTRACT The RpoS sigma factor (also called σ S or σ 38 ) is known to regulate at least 50 genes in response to environmental sources of stress or during entry into stationary phase. Regulation of RpoS abundance and activity is complex, with many factors participating at multiple levels. One factor is the nutritional stress signal ppGpp. The absence of ppGpp blocks or delays the induction of rpoS during entry into stationary phase. Artificially inducing ppGpp, without starvation, is known to induce rpoS during the log phase 25- to 50-fold. Induction of ppGpp is found to have only minor effects on rpoS transcript abundance or on RpoS protein stability; instead, the efficiency of rpoS mRNA translation is increased by ppGpp as judged by both RpoS pulse-labeling and promoter-independent effects on lacZ fusions. DksA is found to affect RpoS abundance in a manner related to ppGpp. Deleting dksA blocks rpoS induction by ppGpp. Overproduction of DksA induces rpoS but not ppGpp. Deleting dks A neither alters regulation of ppGpp in response to amino acid starvation nor nullifies the inhibitory effects of ppGpp on stable RNA synthesis. Although this suggests that dksA is epistatic to ppGpp, inducing ppGpp does not induce DksA. A dksA deletion does display a subset of the same multiple-amino-acid requirements found for ppGpp 0 mutants, but overproducing DksA does not satisfy ppGpp 0 requirements. Sequenced spontaneous extragenic suppressors of dksA polyauxotrophy are frequently the same T563P rpoB allele that suppresses a ppGpp 0 phenotype. We propose that DksA functions downstream of ppGpp but indirectly regulates rpoS induction.

rpoS

Stringent response

Sigma factor

Overproduction

10.1128/jb.184.16.4455-4465.2002

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Citations (190)

Purification and some characteristics of an oestrogen sulphotransferase from guinea pig adrenal gland and its non-identity with adrenal pregnenolone sulphotransferase

Biochemical Journal (1990)

R. Hobkirk Mary Ann Glasier Larissa Brown

An oestrogen sulphotransferase, active towards both oestrone and oestradiol, and of high specific activity, is present in cytosol prepared from adrenal glands of both sexes of English Shorthair and Hartley guinea pigs. The ovarian and testicular cytosolic activities of this enzyme are markedly low in comparison with the adrenal activity. The adrenal enzyme is distinct from an accompanying pregnenolone sulphotransferase as judged by f.p.l.c. gel filtration, chromatofocusing, and differences in activation brought about by the addition of thiol groups. The oestrogen sulphotransferase behaved as a 67 kDa protein on a Sephadex G100 column and as a 48 kDa protein on f.p.l.c. gel-filtration columns. Two forms of the enzyme with apparent pI values of 6.1 and 5.5 were eluted during f.p.l.c. chromatofocusing. Sequential salt fractionation, f.p.l.c. gel filtration and elution from an agarose-hexane-adenosine-3',5'-diphosphate affinity gel has resulted in a preparation which, when resubmitted to f.p.l.c. gel filtration, yields a considerably purified oestrogen sulphotransferase. When submitted to SDS/polyacrylamide-gel electrophoresis under reducing conditions, a main protein band of 34-36 kDa is observed. It is suggested that the enzyme may exist as a dimer in the cytosol.

10.1042/bj2680759

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Composers' Workshop, May 20, 1999

Scott Ligocki Jennifer Han Laura Routt Larissa Brown Jacob Humphrey

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Intramolecular Regulation of the Opposing (p)ppGpp Catalytic Activities of Rel _Seq , the Rel/Spo Enzyme from Streptococcus equisimilis

Journal of Bacteriology (2002)

Undine Mechold Helen Murphy Larissa Brown Michael Cashel

ABSTRACT Catalytic and regulatory domains of the Rel/Spo homolog of Streptococcus equisimilis affecting (p)ppGpp synthesis and degradation activities have been defined, and opposing activities of the purified protein and its fragments have been compared. Two major domains of the 739-residue Rel Seq protein are defined by limited proteolytic digestion. In vitro assays of the purified N-terminal half-protein reveal synthesis of (p)ppGpp by an ATP-GTP 3′-pyrophosphotransferase as well as an ability to degrade (p)ppGpp by a Mn 2+ -dependent 3′-pyrophosphohydrolase. Removal of the C-terminal half-protein has reciprocal regulatory effects on the activities of the N-terminal half-protein. Compared to the full-length protein, deletion activates (p)ppGpp synthesis specific activity about 12-fold and simultaneously inhibits (p)ppGpp degradation specific activity about 150-fold to shift the balance of the two activities in favor of synthesis. Cellular (p)ppGpp accumulation behavior is consistent with these changes. The bifunctional N-terminal half-protein can be further dissected into overlapping monofunctional subdomains, since purified peptides display either degradation activity (residues 1 to 224) or synthetic activity (residues 79 to 385) in vitro. These assignments can also apply to RelA and SpoT. The ability of Rel Seq to mediate (p)ppGpp accumulation during amino acid starvation in S. equisimilis is absent when the protein is expressed ectopically in Escherichia coli. Fusing the N-terminal half of Rel Seq with the C-terminal domain of RelA creates a chimeric protein that restores the stringent response in E. coli by inhibiting unregulated degradation and restoring regulated synthetic activity. Reciprocal intramolecular regulation of the dual activities may be a general intrinsic feature of Rel/Spo homolog proteins.

Stringent response

10.1128/jb.184.11.2878-2888.2002

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Citations (178)

So Wild a Dream

Larissa Brown

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Promoter Substitution and Deletion Analysis of Upstream Region Required for rpoS Translational Regulation

Journal of Bacteriology (1998)

Christofer Cunning Larissa Brown Thomas Elliott

ABSTRACT The RpoS sigma factor of enteric bacteria is required for the increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to high osmolarity. RpoS is also a virulence factor for several pathogenic species, including Salmonella typhimurium . The activity of RpoS is regulated at both the level of synthesis and protein turnover. Here we investigate the posttranscriptional control of RpoS synthesis by using rpoS-lac protein and operon fusions. Substitution of the native rpoS promoters with the tac or lac UV5 promoters allowed essentially normal regulation after growth into stationary phase in rich medium or after osmotic challenge. Regulation of these fusions required the function of hfq , encoding the RNA-binding protein host factor I (HF-I). Short deletions from the 5′ end of the rpoS transcript did not affect regulation very much; however, a larger deletion mutation that still retains 220 bp upstream of the rpoS ATG codon, including a proposed antisense element inhibitory for rpoS translation, was no longer regulated by HF-I. Several models for regulation of rpoS expression by HF-I are discussed.

rpoS

Sigma factor

10.1128/jb.180.17.4564-4570.1998

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Citations (59)