Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Animal model of acute pericarditis and its progression to pericardial fibrosis and adhesions: ultrastructural studies

To study the evolution of pericardial inflammation, we have developed a model of pericarditis in sheep by surgically injecting heat-killed staphylococci and Freund's adjuvant into the pericardial cavity under sterile conditions. The pericarditis evolved through the following phases: 1) inflammatory response, 2) mesothelial cell injury and desquamation, and 3) fibrotic phase. At 3-24 hr there was increased microvascular permeability, which resulted in the exudation of fluid, neutrophils, macrophages, and fibrin into the pericardial cavity and the pericardial interstitium. By 72 hr, large numbers of inflammatory cells were aggregated on the mesothelial surfaces and dispersed throughout the pericardial cavity, either as free-floating cells or located between strands of fibrin. At 6 days, fibrinolysis was apparent along the mesothelial surfaces; and newly formed collagen fibrils were deposited throughout the interstitial spaces and among the aggregated cells. These fibrils provided a matrix for the growth of new blood and lymphatic vessels into new connective tissue on both parietal and visceral pericardial surfaces. At 2 weeks, intrapericardial fibrosis had produced focal adhesions between the pericardial surfaces. At 1 month, extensive areas of the pericardial cavity were obliterated. By 9 months, there was a marked reduction in the numbers of cells and blood vessels and increased deposition of collagen and elastic fibers. The intrapericardial injection of heat-killed staphylococci and adjuvant provides a reproducible animal model to study the time course of pericardial inflammation.     

Dominant expression of an amino-acid uptake deficiency in carrot somatic fusion hybrids

Somatic hybrids were selected previously by their ability to grow in medium containing normally inhibitory levels of the two amino acid analogs aminoethylcysteine (AEC) and 5-methyltryptophan (5MT) following fusion of protoplasts from a cell strain resistant to AEC with protoplasts resistant to 5MT. The hybrid nature of the selected clones was shown by several criteria including the presence of another resistance, azetidine-2-carboxylate (A2C), carried by one of the parental strains which was not selected for in the initial hybrid selection scheme. The characterization presented here shows that the AEC resistance in the parental strain, as well as the two somatic hybrids, was due to decreased AEC uptake. Also the 5MT resistance in the hybrids, as in the parent was caused by a feedback altered form of the tryptophan biosynthetic control enzyme, anthranilate synthase which leads to increases in free tryptophan. The A2C resistance was caused by the accumulation of free proline by a mechanism which has not been studied. These studies confirm that AEC resistance caused by decreased uptake can be expressed dominantly in protoplast fusion hybrids.Abbreviations A2C Azetidine-2-carboxylate - AEC Aminoethylcysteine - 5MT 5-methyltryptophan     

Influence of Nitrogen Fertilizers on Growth, Spore Production and Germination, and Biocontrol Potential of Trichoderma and Gliocladium

The mycelial weight of eight out of nine isolates of Trichoderma spp. and Gliocladium virens increased in media supplemented with 2000 mg/l of nitrogen (N) from the fertilizers NH₄Cl, NaNO₃, and a commercial 20–20–20. In general, the greatest increase in growth (up to 311 %) occurred with 20–20–20. The extent of growth was similar with either NH₄Cl or NaNO₃, but was less than that with 20–20–20. Measured by radial development on agar surfacesgrowth of isolates was either not affected or was constricted by supplemental fertilizers. Production of conidia by six out of eight isolates was stimulated by 20–20–20 but not by NH₄Cl or NaNO₃. Germination of conidia of all isolates, generally was high (> 85 %) on amended and nonamended agar. Chlamydospore formation by three Trichoderma isolates in liquid media was not affected by fertilizers. Antagonism or overgrowth of the pathogen Rhizoctonia solani by Trichoderma isolates in culture was reduced appreciably by NaNO₃, but was not affected by NH₄Cl or 20–20–20. Addition of 20–20–20 to natural soil did not reduce further the survival of R. solani caused by germling preparations of six out of seven Trichoderma isolates. However, reduction in survival of the pathogen caused by a T. hamatum isolate was stimulated further (45 %) by the fertilizer.     

Clinical hypersensitivity to specific aerosol challenge in parenterally immunized calves.

This paper reports an experiment designed to demonstrate that the calf lung can be sensitized to a specific respirable challenge following parenteral immunization with a nonliving antigen (human serum albumin). The possibility that immune-mediated injury could subsequently interfere with nonspecific mucosal defenses was also investigated by infecting calves with Pasteurella haemolytica after the antigen challenge and assessing pulmonary clearance of the organism. The results indicated that specific aerosol challenge produces reversible signs of respiratory hypersensitivity and that persistence of incidental infection in the upper respiratory tract is potentiated. Since the calves were sensitized by an immunization regime which imitated conventional vaccination, this study highlights the potential dangers of inactivated parenteral respiratory vaccines.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916.

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

Superoxide removal and radiation protection in bacteria

Previous work with procaryotic cells has identified one kind of lethal damage from ionizing radiation which occurs only within a specific range of low O2 concentrations, about 10(-6) to 10(-4) M. Within this range, protection can occur in three ways: through the enzymatic decomposition of hydrogen peroxide (H2O2) by added catalase, through the enzymatic degradation of superoxide anion radicals (.O2-) by added superoxide dismutase (SOD), and through scavenging hydroxyl radicals (.OH) by various additives. These results indicate that three radiolytic products, H2O2, .OH, and .O2- (and/or the conjugate acid, the perhydroxyl radical, .HO2) are involved in this single kind of radiation-induced damage. Although the radiolytic productions of H2O2 and .O2- are strongly enhanced in higher O2 concentrations, neither enzyme protects when these air-equilibrated bacteria are irradiated. These experiments address this apparent contradiction and focus on the specific issue of why the addition of SOD protects at low but not at high O2 concentrations. We propose that, at a given O2 concentration, .O2- (and/or .HO2) may either react (with some cellular component?) to cause damage or react (with itself) to form hydrogen peroxide (H2O2). The specific O2 concentration during irradiation would determine the relative rates of these competing reactions and therefore the O2 concentration itself would establish whether or not we will observe damage from .O2-.