Nature of stress on the atomic level in dense polymer systems.

Stress in dense polymer systems is classically viewed as being molecular in character and is based on the entropic spring concept. A description on the atomic level has been developed on the basis of extensive computer simulations. An important new concept is the intrinsic monomer stress (IMS), the individual monomer contribution to the macroscopic stress referred to a local moving coordinate system in which the backbone bonds attached to that monomer are fixed. The IMS is time-independent and, for a given polymer system at fixed density, has the same value in the equilibrium melt, with the melt undergoing stress relaxation, and in the deformed cross-linked system.

Evaluation of transmembrane helix prediction methods using the recently defined NMR structures of the coat proteins from bacteriophages M13 and Pf1.

Currently, there are a large number of hydropathy scales available to predict the presence of transmembrane segments within integral membrane proteins. These scales and their subsequent numerical manipulations provide an aid in the determination of topology in transmembrane proteins. In order to analyse the accuracy of these procedures to correctly identify the boundaries of a transmembrane segment, 13 methods were applied to the amino-acid sequence of the coat proteins from the bacteriophages Pf1 and M13. These monotopic integral membrane proteins have been incorporated into detergent micelles and their structures have recently been solved using NMR. The predicted regions were then compared to their NMR-determined structures. All methods used were able to detect a transmembrane region within the protein sequence. However, there was considerable differences in their accuracy in determining the boundaries of the main transmembrane alpha-helix. Surprisingly, the methods which worked the best for Pf1 coat protein had poor accuracy in identifying the transmembrane region correctly in the M13 protein. It was concluded that a number of methods should be utilized in order to obtain a clear model of transmembrane protein topology, and that regardless of how closely related two proteins are, a different conclusion may be obtained from different prediction procedures.

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster.

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase.

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.

Zidovudine and dideoxynucleosides deplete wild-type mitochondrial DNA levels and increase deleted mitochondrial DNA levels in cultured Kearns-Sayre syndrome fibroblasts.

Kearns-Sayre syndrome is the most commonly diagnosed mitochondrial cytopathy and produces severe neuromuscular symptoms. The most frequent cause is a mitochondrial DNA deletion that removes a 4977-base pair segment of DNA that includes several genes encoding for respiratory chain subunits. Treatment of AIDS patients with nucleoside analogs has been reported to cause mtDNA depletion and myopathies. Here, we report that azidothymidine, dideoxyguanosine, and dideoxycytidine cause a depletion of wild-type mtDNA while increasing the levels of deleted mitochondria DNA in Kearns-Sayre syndrome fibroblasts. The result of these effects is a large increase in the relative amounts of delta mtDNA in comparison to wild type mtDNA. We found that Kearns-Sayre syndrome fibroblasts are a mixed population of cells with deleted mtDNA comprising from 0 to over 20% of the total mtDNA in individual cells. Treatment of cloned cell lines with dideoxycytidine did not result in increased levels of delta mtDNA. The results suggest that nucleoside analogs may act to increase the average delta mtDNA levels in a mixed population of cells by preferentially inhibiting the proliferation of cells with little or no delta mtDNA. This raises the possibility that modulation of deleted mtDNA levels may occur by similar mechanisms in vivo, in response to the influence of exogenous agents.

The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death.

Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing. However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions. We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway. The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins. An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB. The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B. The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability. The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome. Although the cloned C. freundii entericidin locus is expressed in E. coli independently of ompR, it carries an additional ompR-like gene called ecnR. The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death.

Nucleotide sequence and overexpression of the tellurite-resistance determinant from the IncHII plasmid pHH1508a.

The transcription and translation of the tellurite-resistance (TeR) genes of the HII incompatibility group plasmid, pHH1508a, were studied. The nucleotide (nt) sequence of the TeR region was determined and two possible open reading frames, tehA and tehB, were identified. The direction of transcription and translation of these genes was confirmed through the preparation of lacZ and phoA (encoding alkaline phosphatase) fusions. The transcription start point was identified in the sequence using RNA primer extension. The tehA gene codes for a 36-kDa polypeptide which is highly hydrophobic. The TehA protein appears to be located in the inner membrane of the bacterial cell since tehA fusions with both phoA and lacZ were obtained and expressed. The tehB gene codes for a 23-kDa polypeptide which appears to be relatively hydrophilic and is probably located in the cytoplasm. Both proteins were overproduced using a T7 RNA polymerase/promoter system. No nt or amino acid sequence homology could be found between this TeR determinant and the TeR genes from the IncHI-2 plasmid, pMER610, and the IncP alpha plasmid, RK2.

Coordinate regulation of murein peptidase activity and AmpC beta-lactamase synthesis in Escherichia coli.

In the periplasmic space of Escherichia coli, the (L)-m-A2pm-(D)-m-A2pm peptide, the lipoprotein, and the AmpC beta-lactamase are controlled by growth rate. To explain this coordinate regulation, it is proposed that the AmpC protein functions as an LD-endopeptidase in addition to its known function as a beta-lactamase. As LD-peptides, DD-peptides and beta-lactams are structurally similar, LD-peptidases may belong to the larger family of DD-peptidases and serine beta-lactamases. In contrast to E. coli, many related bacteria possess an inducible AmpC protein. Several gene systems necessary for AmpC induction are known to affect various aspects of peptidoglycan metabolism. It is proposed that AmpC induction occurs indirectly via a recyclable cell wall peptide.

Quantification of mitochondrial DNA in heteroplasmic fibroblasts with competitive PCR.

Kearns-Sayre syndrome (KSS) is a disease with severe clinical symptoms that often arises from a mitochondrial DNA deletion of 4977 bp. Quantification of defective mitochondrial DNA is important since the severity of symptoms in KSS is thought to be related to increased content of abnormal mitochondrial DNA. We developed a rapid, quantitative and competitive PCR assay to measure both wild-type and mutant forms of mitochondrial DNA in cells from KSS patients. The assay can accurately measure absolute numbers of mitochondrial DNA per cell by normalizing to a single copy nuclear gene.

Complementation of growth defect in an ampC deletion mutant of Escherichia coli.

beta-Lactamase genes of class-A (Rtem) and class-C (ampC) were placed under control of an inducible tac-promoter and expressed in Escherichia coli. Expression of RTEM had no observable effect on the growth properties of E. coli strains HB101 (ampC+) or MI1443 (delta ampC). E. coli MI1443 exhibited a decline in growth rate at mid-exponential phase which could be delayed by expression of AmpC at early-exponential phase. AmpC expression otherwise inhibited growth, particularly during the transition into exponential phase where growth was prevented altogether. We suggest that the AmpC beta-lactamase, but not RTEM, may have an additional cellular function as a peptidoglycan hydrolase.

The fumarate and dimethylsulphoxide reductases of anaerobic electron transport inEscherichia coli: current status and future perspectives.

CONCLUSION: Research on bacterial anaerobic electron transport is at a crossroads. The introduction of molecular cloning, expression and mutagenesis techniques are providing answers to many long-standing problems and our understanding of structure-function relationships is progressing rapidly. Future application of emerging biophysical techniques together with progress in crystallography promises to provide answers to our current questions.

Topological characterization of Escherichia coli DMSO reductase by electron paramagnetic resonance spectroscopy of an engineered [3Fe-4S] cluster.

We have applied the technique of distance estimations using the exogenous paramagnetic probe dysprosium (III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase (DmsABC) in situ in cytoplasmic membrane and whole cell preparations. The electron transfer subunit (DmsB) of this enzyme contains four [4Fe-4S] clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes. We have utilized a mutant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been changed to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) Biochemistry 30, 8296-8305]. This mutant (DmsB-C102S) has a single magnetically isolated EPR visible [3Fe-4S] cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe. We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhances the spin relaxation of the [3Fe-4S] signal in everted membrane vesicles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane. We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.