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The Escherichia coli proU operon encodes a high-affinity, binding-protein-dependent transport system for the osmoprotectant glycine betaine. Expression of proU is osmoregulated, and transcription of this operon is greatly increased In cells grown at high osmolarity. Characterization of the proU operon and its promoter provided results similar to those published elsewhere (Gowrishankar, 1989; Stirling et al., 1989). The previously identified proU601 mutation, which leads to increased proU expression both at low- and high osmolarity, is a G to A transition in the Pribnow box of the proU promoter, which increases the homology of the -10 region to the consensus sequence of E. coli promoters. Using an antiserum raised against a ProV-β-gaiactosidase hybrid protein, we have identified ProV as a protein associated with the cytoplasmic membrane. This cellular location is consistent with its proposed role as the energy-coupling component of the ProU transport system. [ABSTRACT FROM AUTHOR]

The proU locus of Escherichia coli encodes a high-affinity, binding-protein-dependent transport system (ProU) for the osmoprotectant glycine betaine. We cloned this locus into both low-copy-number lambda vectors and multicopy plasmids and demonstrated that these clones restore osmotically controlled synthesis of the periplasmic glycine betaine binding protein (GBBP) and the transport of glycine betaine in a Δ(proU) strain. These clones allowed us to investigate the influence of osmolarity on ProU transport activity independent of the osmotically controlled expression of proU. ProU activity was strongly stimulated by a moderate increase in osmolarity and was partially inhibited by high osmolarity. This activity profile differs from the profile of the osmotically regulated proU expression. The proU locus is organized in an operon and the position of the structural gene (proV) for GBBP is defined using a minicell system. We determined that at least three proteins (in addition to GBBP) are encoded by the proU locus. We also investigated the permeation of glycine betaine across the outer membrane. At low substrate concentration (0.7 μM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins. [ABSTRACT FROM AUTHOR]

The envelope glycoproteins of Rubella virus, E1 and E2, mediate cell tropism, and E1 in particular plays a pivotal role in the fusion of the virus with the endosomal membrane. Both are the prime targets of the humoral immune response. Recombinant variants of the E1 ectodomain as well as E1 antigen preparations from virus lysates are commonly used to detect anti-Rubella immunoglobulins in human sera. Hitherto, recombinant E1 for diagnostic applications has been produced chiefly in eukaryotic expression systems. Here, we report the high-yield overproduction of an engineered E1 ectodomain in the Escherichia coli cytosol and its simple and convenient renaturation into a highly soluble and immunoreactive conformation. C-Terminal fusion to one or two units of the E. coli chaperone SlyD enhances expression, facilitates in vitro refolding, and improves the overall solubility of Rubella E1. As part of this fusion protein, the E1 ectodomain fragment of residues 201-432 adopts an immunoreactive fold, providing a promising tool for the sensitive and specific detection of anti-E1 IgG in Rubella serology. Two disulfide bonds in the membrane-adjacent part of the E1 ectodomain are sufficient to generate conformations with a high and specific antigenicity. The covalently attached chaperone modules do not impair antibody recognition and binding of Rubella E1 when assessed in a heterogeneous immunoassay. SlyD and related folding helpers are apparently generic tools for the expression and refolding of otherwise unavailable proteins of diagnostic or medical importance.

The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.

Background: Different subtypes of HIV-1 are prevalent in various geographical regions. Knowledge of their distribution is of importance with respect to possible differences in biological properties (such as reported for subtype E) as well as to diagnostic problems that may arise when specific subtypes are not recognized by standard serological assays., Objectives: The objectives of this study were to investigate the presence of the five major subtypes of HIV-1 (A-E) in the Austrian population and to estimate the prevalence of the individual subtypes in different risk groups., Study Design: Serum samples from 88 HIV-1 positive patients were tested for the presence of subtype-specific antibodies using a peptide ELISA., Results: The majority of the patients were shown to be infected with HIV-1 subtype B, but infections with subtypes A, C, and E were also detected in the Austrian population, primarily in the heterosexual transmission group. While subtypes A and C were probably imported from different African countries, subtype E appears to have been introduced by sex tourists returning from Thailand., Conclusion: Introduction of HIV subtypes other than B from Africa and Asia into Austria has already occurred and will certainly increase within the next few years.

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a KD of 5.2 microM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (KD of 1.4 microM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.

Glycine 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 microM. 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 lambda plac Mu-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 phi(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.