Merna Villarejo

Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12

SummaryGlycine betaine, which functions as an osmoprotectant, is accumulated to high intracellular concentrations in Escherichia coli at high osmolarity. We demonstrate the presence of a high-affinity, binding protein dependent transport system for glycine betaine, which is encoded by the proU region. We show the osmotically regulated synthesis of a 32 kDa periplasmic protein that is a glycine betaine binding protein with a KD of 1.4 μM. ProU-mediated glycine betaine transport is osmotically stimulated at the level of gene expression. The osmolarity of the medium also regulates the activity of the transport system, while binding of glycine betaine to its binding protein is independent of the osmolarity. We also find a second glycine betaine transport system that is dependent on proP and exhibits a lower substrate affinity. Like ProU, this system is regulated at two levels: both gene expression and the activity of the transport system are osmotically stimulated. Using λplacMu-generated lacZ operon and gene fusions, we find that expression of the proU region is osmotically regulated at the level of transcription. We cloned a part of the proU region together with the Φ(proU-lacZ)hyb2 gene fusion into a multicopy plasmid and show that the DNA sequences required in cis for osmotic regulation are present on the plasmid.

Importance of Undergraduate Research for Minority Persistence and Achievement in Biology

This study examines the association between undergraduate research participation and college success in the biological sciences. We find that undergraduate research participation is significantly associated with earning a baccalaureate degree and with persistence and outstanding performance among biology majors for all racial/ethnic groups at a large research university.

Promoter selectivity control of Escherichia coli RNA polymerase by ionic strength: differential recognition of osmoregulated promoters by EσD and EσS holoenzymes

Transcription in vitro of two osmoregulated promoters, for the Escherichia coli osmB and osmY genes, was analysed using two species of RNA polymerase holoenzyme reconstituted from purified core enzyme and either σD (σ70, the major σ in exponentially growing cells) or σS (σ38, the principal σ at stationary growth phase). Under conditions of low ionic strength, the osmB and osmY promoters were transcribed by both EσD and EσS. Addition of up to 400 mM potassium glutamate (K glutamate), mimicking the intracellular ionic conditions under hyper‐osmotic stress, specifically enhanced transcription at these promoters by EσS but inhibited that by EσD. At similar high concentrations of potassium chloride (KCI), however, initiation at both these promoters was virtually undetectable. These data suggest that the RNA polymerase, EσS, itself can sense osmotic stress by responding to changes in intracellular K glutamate concentrations and altering its promoter selectivity in order to recognize certain osmoregulated promoters.

Structural and catalytic properties of peptidase N from Escherichia coli K-12

Abstract Escherichia coli K-12 strains contain a single cytosolic activity, peptidase N, capable of hydrolyzing alanine-p-nitroanilide. The enzyme is a monomeric, acidic protein with a molecular weight of 87,000, containing a sulfhydryl group essential for activity. Substrate specificity studies show a preference for hydrophobic peptides and a requirement for free α-amino groups. Peptidase N has no measurable endoproteolytic activity on protein, large peptide, or signal peptide substrates. These results suggest that peptidase N is a hydrophobic specificity aminopeptidase whose physiological role is as yet still unclear. It is likely to be involved in the late stages of protein turnover events or in degradation of specific cellular components such as attenuator peptides.

Regulation of gene expression in Escherichia coli by the local anesthetic procaine

Procaine treatment of phoA-lac, malE-lac and ompF-lac operon fusion strains results in reduced β-galactosidase levels, suggesting that procaine inhibits transcription of these target genes.

Evidence for two lac Y gene derived protein products in the E.coli membrane

Summary The membrane protein composition of several lac operon mutant strains has been determined by amino acid double labeling and analysis by SDS-PAGE or gel filtration in SDS. A single component of apparent MW = 30,000 could be identified as the product of the wild type lac Y gene when membrane proteins were dissolved in SDS at room temperature. Heating in SDS in the presence of 50 MM DTT or iodoacetamide allowed complete denaturation and resolution of two protein components with MWs of 33,000 and 15,000. Both polypeptides were altered in strains with mutations in lac Y . The two polypeptides from a wild-type strain fractionated as a single component during extraction and purification in the non-denaturing detergent, Triton X-100. The sum of the MWs of the two proteins is approximately equal to the MW of the protein product predicted from the lac Y gene DNA sequence.

Localization of the lactose permease protein(s) in the E.coli envelope

The amino acid double labeling technique was used to identify and localize membrane-bound lactose operon proteins in E.coli. Both the “M” protein, thought to be the y gene product, and a polypeptide of MW ∼15,000 appeared in the membrane following lac operon induction. The amounts of these two proteins were approximately equal. The inner and outer membrane layers of the cell envelope were separated by sucrose density gradient centrifugation or by selective solubilization of inner membranes with the detergent Sarkosyl. When gentle lysis conditions were employed to prepare membrane vesicles, both lac induced proteins fractionated with the inner membrane. However, the “M” protein was more easily randomized in the envelope structure by sonication than the 15,000 dalton component or an inner membrane marker enzyme.

Monoclonal antibody to an integral membrane protein, the lactose permease☆

A monoclonal IgG antibody directed against the lactose permease was produced from animals inoculated with membranes of a lac Y plasmid strain. The appropriate antibody was selected by a series of ELISA assays in which membranes, purified permease, or a lac Y-Z chimeric protein was the immobilized antigen. The antibody recognizes a portion of the permease exposed on the surface of membrane vesicles but does not inhibit lactose transport.