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Biomedical subjects

E R Olson

Publications and source records attributed to E R Olson.

At least 19 recordsLinked to original sources
PubMed2000

Escherichia coli requires the protease activity of FtsH for growth.

FtsH protease, the product of the essential ftsH gene, is a membrane-bound ATP-dependent metalloprotease of Escherichia coli that has been shown to be involved in the rapid turnover of key proteins, secretion of proteins into and through the membrane, and mRNA decay. The pleiotropic effects of ftsH mutants have led to the suggestion that FtsH possesses an ATP-dependent chaperone function that is independent of its protease function. When considering FtsH as a target for novel antibacterials, it is necessary to determine which of these functions is critical for the growth and survival of bacteria. To address this, we constructed the FtsH mutants E418Q, which retains significant ATPaseactivity but lacks protease activity, and K201N, which lacks both protease and ATPase activities. These mutants were introduced into an E. coli ftsH knockout strain which has wild-type FtsH supplied from a plasmid under control of the inducible araBAD promoter. Since neither mutant would complement the ftsH defect produced in the absence of arabinose, we conclude that the protease function of FtsH is required for bacterial growth.

ATP-Dependent Proteases↗
PubMed2000

Baseline susceptibility to imidacloprid and cross resistance patterns in Colorado potato beetle (Coleoptera: Chrysomelidae) populations.

During 1995-1998, we tested 134 geographically discrete populations of Colorado potato beetle, Leptinotarsa decemlineata (Say), from the United States, Canada, Germany, France, and Poland for susceptibility to imidacloprid. Neonates were assayed on potato-based agar diet incorporated with imidacloprid and exposed on filter paper to esfenvalerate, azinphosmethyl, and carbofuran to characterize cross-resistance. In all 4 yr, Long Island populations were the most tolerant to imidacloprid, with LC50s ranging up to 29 times higher than the most susceptible populations. Responses to imidacloprid did not change significantly on farms where populations were assayed over time, except for those from Long Island, which doubled in overall tolerance to imidacloprid since 1995. Much of this tolerance was already present before imidacloprid was used on Long Island. Correlative analysis of the populations tested over the 4 yr indicated positive cross-resistance patterns with esfenvalerate and azinphosmethyl. This response was probably caused by preexisting metabolic and excretion mechanisms selected by previous exposure. There was no significant pattern of cross-resistance with carbofuran or bensultap. Regression slopes were also significantly negatively correlated with LC50 values for imidacloprid, indicating higher heterogeneity, which could lead in further resistance development. We discuss the relative sensitivity of diet-incorporated assays with neonates compared with other bioassay studies. Based on a pooled group of susceptible populations tested in 1995, a baseline LC50 of 0.39 ppm and a discriminating concentration of 8 ppm were suggested to detect early stages of resistance in "suspect" populations. We also suggest application strategies for imidacloprid that reduce selection pressure.

Animals↗
PubMed2000

Novel streptopyrroles from Streptomyces rimosus with bacterial protein histidine kinase inhibitory and antimicrobial activities.

A series of halogenated pyrrolo [2,1-b] [1,3] benzoxazines (1 approximately 9) was isolated from fermentations of an actinomycete strain X10/78/978 (NCIMB40808), identified as Streptomyces rimosus, during a microbial extract screening programme to identify inhibitors of bacterial histidine kinase. The structures of these compounds were elucidated by spectroscopic methods including the HMQC, HMBC and INADEQUATE NMR experiments. The structure of 1 was confirmed by X-ray crystallographic studies. Compounds 5 and 6 were produced in fermentations in the presence of NaBr and NaI respectively. The most abundant member of the series, streptopyrrole, 1, inhibited the nitrogen regulator II (NRII) histidine kinase from Escherichia coli with an IC50 of 20 microM and exhibited antimicrobial activity against a range of bacteria and fungi.

Anti-Bacterial Agents↗
PubMed1999

Functional and biochemical characterization of Escherichia coli sugar efflux transporters.

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogue o-nitrophenyl-beta-D-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.

Biological Transport↗
PubMed1999

The identification of a new family of sugar efflux pumps in Escherichia coli.

Using a functional cloning strategy with an Escherichia coli genomic plasmid library, we have identified a new family of sugar efflux proteins with three highly homologous members in the E. coli genome. In addition, two open reading frames, one present in Yersinia pestis and the other in Deinococcus radiodurans, appear to encode closely related proteins. An in vitro transport assay using inside-out membrane vesicles prepared from overproducing strains was used to demonstrate that members of this new family can efflux [14C]-lactose. As sugar efflux phenomena have been reported previously in several bacterial species including E. coli, the identification of a new family of sugar efflux proteins may help to reveal the physiological role of sugar efflux in metabolism. It is proposed that the E. coli members of this family, whose functions were previously unknown, be given the gene family designation SET for sugar efflux transporter.

Antiporters↗
PubMed1997

Escherichia coli proteome analysis using the gene-protein database.

The gene-protein database of Escherichia coli is a collection of data, largely generated from the separation of complex mixtures of cellular proteins on two-dimensional (2-D) polyacrylamide gel electrophoresis. The database currently contains about 1600 protein spots. The data are comprised of both identification information for many of these proteins and data on how the level or synthesis rates of proteins vary under different growth conditions. Three projects are underway to further elucidate the E. coli proteome including a project to localize on 2-D gels all of the open reading framed encoded by the E. coli chromosome, a project to determine the condition(s) under which each open reading frame is expressed and a project to determine the abundance and location of each protein in the cell. Applications for proteome databases for cell modeling are discussed and examples of applications in therapeutic drug discovery are given.

Bacterial Proteins↗
PubMed1996

Global analysis of proteins synthesized during phosphorus restriction in Escherichia coli.

The pattern of proteins synthesized in Escherichia coli during steady-state growth in media with ample inorganic phosphate (Pi), upon limitation for Pi (without an alternative phosphorous compound), and during steady-state growth in media containing phosphonate (PHN) as the sole P source was examined by two-dimensional gel electrophoresis. Of 816 proteins monitored in these experiments, all those with differential synthesis rates greater than 2.0 or less than 0.5 upon phosphate limitation (P limitation) or during growth on PHN compared with their rates in the cultures with Pi were classified as belonging to the PL or PHN stimulon, respectively. The PL stimulon included 413 proteins, 208 showing induced synthesis and 205 showing repressed synthesis. The PHN stimulon was smaller: it included 257 proteins; 227 showed induced synthesis and 30 showed repressed synthesis. The overlap of the two stimulons included 137 proteins: most (118) were ones showing induced synthesis. The promoter regions of genes for several of the proteins with induced or repressed synthesis contained sequences which resembled the consensus sequence for PhoB binding. The aggregate mass of proteins responding to P limitation or growth on PHN was 30 to 40% of the cells' total mass. By comparing the proteins responding to P limitation with those responding to growth on PHN, one can speculate which proteins are likely involved in adapting cells to new P sources or in preparing cells to survive stationary phase.

Bacterial Proteins↗
PubMed1996

Kinetics of expression of the Escherichia coli cad operon as a function of pH and lysine.

The Escherichia coli cadBA genes are regulated at the transcriptional level by external pH and lysine. The membrane-localized CadC protein is required for activation of this operon under inducing conditions, which include acidic external pH, lysine, and oxygen limitation. To better understand the mechanism by which CadC functions, the kinetics of cadBA expression as a function of pH and lysine were examined. By primer extension assays, cadBA expression was detected within 4 min following exposure of cells to one of the inducing stimuli (low pH or lysine), provided that the cells had first been grown to steady state in the presence of the other inducing stimulus. The induction time was three to four times longer when both inducing stimuli were added simultaneously. cadBA expression was shut off within 4 min following a shift from acidic to neutral pH. Treatment of cells with chloramphenicol prevented induction by acidic pH and lysine. Transcription of lysP (encodes a lysine transporter) was also examined, since it is a negative regulator of cadBA expression in the absence of lysine. lysP expression was repressed by lysine but not influenced by pH. Putative transcription start sites for lysP and cadC were determined. Together, these data suggest that CadC senses the lysine- and pH-induced signals separately and that one of the roles of lysine in inducing cadBA may be to repress expression of lysP, thus eliminating the repressing effects of LysP.

Amino Acid Transport Systems↗
PubMed1995

Application of two-dimensional protein gels in biotechnology.

The optimal use of biological systems for technologically developed products will not be achieved until biological systems are completely defined in biochemical terms. Two-dimensional polyacrylamide gel electrophoresis, 2-D gels, are contributing to this goal. These gels separate complex mixtures of proteins into individual polypeptide species. The ultimate use of 2-D gels is the construction of cellular 2-D gel databases which identify the proteins on the gels and catalog their responses to different environmental conditions. In addition to these global analyses, many applications for 2-D gels in basic, applied and clinical research have been shown.

Amino Acid Sequence↗
PubMed1994

Altered pH and lysine signalling mutants of cadC, a gene encoding a membrane-bound transcriptional activator of the Escherichia coli cadBA operon.

The Escherichia coli CadC protein is required for activation of cadBA transcription under conditions of low external pH and exogenous lysine. cadBA encodes proteins involved in the decarboxylation of lysine to cadaverine, and cadaverine excretion. Sequence analysis suggested that CadC contains a single transmembrane segment separating a DNA-binding domain in the amino terminus from a periplasmic domain. Western analysis of subcellular fractions demonstrated that CadC is expressed and concentrated in the cytoplasmic membrane in cells grown either at an inducing pH (pH 5.8) or at a non-inducing pH (pH 7.6). Eight cadC mutants were isolated based on their ability to confer expression of a cadA-lacZ fusion independent of low external pH or exogenous lysine. Five of these mutants expressed the cadA-lacZ fusion at both pH 5.8 and pH 7.6, but retained the requirement for the lysine signal while the other three mutants displayed pH independence at pH 5.8 but not at pH 7.6. These results support a model in which CadC is a membrane-bound transcriptional activator of the cadBA operon capable of sensing (directly or indirectly) signals generated outside the cytoplasmic membrane as a consequence of acidic pH and lysine.

Bacterial Proteins↗
PubMed1994

Roles of LysP and CadC in mediating the lysine requirement for acid induction of the Escherichia coli cad operon.

Expression of the Escherichia coli cadBA operon, encoding functions required for the conversion of lysine to cadaverine and for cadaverine excretion, requires at least two extracellular signals: low pH and a high concentration of lysine. To better understand the nature of the lysine-dependent signal, mutants were isolated which expressed a cadA-lacZ transcription fusion in the absence of lysine while retaining pH regulation. The responsible mutation in one of these isolates (EP310) was in cadC, a gene encoding a function necessary for transcriptional activation of cadBA. This mutation (cadC310) is in a part of the gene encoding the periplasmic domain of CadC and results in an Arg-to-Cys change at position 265, indicating that this part of the protein is involved in responding to the presence of lysine. Three other mutants had mutations mapping in or near lysP (cadR), a gene encoding a lysine transport protein that has previously been shown to regulate cadA expression. One of these mutations is an insertion in the lysP coding region. Thus, in the absence of exogenous lysine, LysP is a negative regulator of cadBA expression. Negative regulation by LysP was further demonstrated by showing that lysP expression from a high-copy-number plasmid rendered cadA-lacZ uninducible. Expression of cadA-lacZ in a strain carrying the cadC310 allele, however, was not affected by the plasmid-expressed lysP. Cadaverine was shown to inhibit expression of the cadA-lacZ fusion in cadC+ cells but not in a cadC310 background.

Acids↗
PubMed1993

Influence of pH on bacterial gene expression.

Bacteria respond to changes in internal and external pH by adjusting the activity and synthesis of proteins associated with many different processes, including proton translocation, amino acid degradation, adaptation to acidic or basic conditions and virulence. While, for many of these examples, the physiological and biological consequence of the pH-induced response is clear, the mechanism by which the transcription/translation machinery is signalled is not. These examples are discussed along with several others in which the function of the gene or protein remains a mystery.

Adaptation, Physiological↗
PubMed1992

Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH.

Expression of the lysine decarboxylase gene (cadA) of Escherichia coli is induced upon external acidification. To dissect the molecular mechanisms responsible for this regulation, we analyzed a 4.2-kbp region upstream from cadA. DNA sequencing revealed two long open reading frames upstream of and on the same strand as cadA. One of these, cadB, is 444 codons long and is situated immediately upstream of cadA. Transcriptional fusions between fragments upstream of cadA and lacZ, Northern (RNA) hybridization, primer extension, and site-directed mutagenesis experiments defined a promoter, Pcad, upstream of cadB that was responsible for pH-regulated expression of cadA. Upstream of Pcad is an open reading frame, cadC, consisting of 512 codons. The predicted amino terminal region of the cadC gene product (CadC) resembles the carboxy-terminal domain of prokaryotic transcriptional activators involved in environmental sensing. Tn10 insertions within or immediately upstream of cadC abolished Pcad activity, suggesting that cadC encodes a positive transcription factor. Expression of plasmid-borne cadC in the Tn10 mutants restored Pcad activity, while introduction of a plasmid expressing truncated CadC resulted in the inability to complement. The presence of Pcad on a multicopy plasmid was found to lower expression arising from chromosomal Pcad, suggesting that a positive-acting factor is limiting. Our data suggests that cadA, cadB, and the acid-inducible Pcad comprise, at least in part, the cad operon which is under control of the cadC product.

Amino Acid Sequence↗
PubMed1991

Chronic postoperative endophthalmitis associated with Actinomyces species.

Actinomyces species, gram-positive, non-spore-forming anaerobic bacilli were isolated from intraocular fluid obtained from four otherwise healthy patients with a delayed onset of postoperative endophthalmitis. One patient had a mixed anaerobic infection with recovery of both Actinomyces israelii and Propionibacterium acnes. In all four patients, early postoperative visual acuity was good but was eventually markedly reduced by intraocular inflammation that was first observed between 21 days and 4 months following uneventful extracapsular cataract extraction and posterior chamber intraocular lens implantation. Inflammation was characterized by anterior segment and vitreous cellular debris in all cases. All eyes responded to therapy that included intraocular, topical, and systemic antibiotics as well as pars plana vitrectomy and partial iridectomy. These cases further illustrate the need for microbiologic investigation, including anaerobic cultures, in all cases of chronic postoperative inflammation following extracapsular cataract extraction, regardless of the time of onset.

Actinomyces↗
PubMed1991

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein.

We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport. dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA). Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein. Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source. dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase. An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly. Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal.

Amino Acid Sequence↗
PubMed1990

Point mutations in a pBR322-based expression plasmid resulting in increased synthesis of bovine growth hormone in Escherichia coli.

The bGH cDNA coding for bovine growth hormone (bGH) is expressed poorly in Escherichia coli using a pBR322-based expression plasmid. Random mutagenesis of the plasmid gave rise to two types of plasmid mutants which increased the expression of bGH. One class had single base changes in the first four codons of the bGH sequence. The second class had single base changes in regions of the plasmid involved in controlling plasmid replication but had little effect on plasmid copy number.

Amino Acid Sequence↗