Minicell production and bacteriophage superinducibility of thymidine-requiring strains of Haemophilus influenzae.

Aminopterin- or trimethoprin-resistant thymidine-requiring strains of Haemophilus influenzae produce minicells, and the ratio of minicells to cells increases during the stationary phase of growth. Strain LB11, isolated after mutagenesis of a thymidine-requiring strain (Rd thd), produces more minicells than the parent strain. The mutations involved in high frequency minicell production have been transferred into the wild type (strain Rd) by transformation. The thymidine requirement in the resulting strain, MCl, is essential for minicell production, since spontaneous revertants of MCl to prototrophy do not produce minicells. The ratio of minicells to cells was increased more than 10(3)-fold by differential centrifugation. The minicells contain little or no deoxyribonucleic acid (DNA). Phage HPlcl apparently cannot attach to minicells. Competent cells of LB11 and its thymidine-requiring parent strain produce defective phage as a result of exposure to transforming DNA, whereas only LB11 produces many defective phage in response to the competence regime alone. Competent HP1c1 and S2 lysogens of MC1 and Rd thd are also superinducible by transforming DNA, but competent LB11 lysogens produced about the same amount of HP1c1 or S2 phage with or without exposure to transforming DNA possibly because of competition between the induced defective phage and Hp1c1 or S2 phage.     

Genetic and kinetic evidence for different types of postreplication repair in Escherichia coli B.

The changes in molecular weight of deoxyribonucleic acid (DNA) synthesized after ultraviolte irradiation of Escherichia coli WP28 uvrA, and strains additionally mutant at polA, exrA, recA, and exrA and polA loci, were examined by alkaline sucrose gradient centrifugation. In a repari=deficient uvrA recA strain, the frequency of breaks in newly synthesized DNA was equal to that for pyrimidine dimers in parental DNA. Measurements of the amounts and rates of postreplication repair of these breaks indicate that (i) repair is two to three times faster when DNA polymerase I is present, although (ii) almost all breaks are repaired regardless of DNA polymerase I activity. (iii) Increased ultraviolet doses lead to an increase in the proportion of breaks remaining unrepaired in uvrA recA, UVRA exrA, and uvrA exrA polA strains. The numbers of unrepaired breaks resemble the numbers expected if repair of one lesion is prevented by proximity of a second lesion.     

Effect of Photoreactivation on the Filling of Gaps in Deoxyribonucleic Acid Synthesized After Exposure of Escherichia coli to Ultraviolet Light

Daughter-strand gaps in deoxyribonucleic acid (DNA) synthesized after exposure of excision-deficient Escherichia coli to ultraviolet light are filled during subsequent incubation in buffer, and the rate of filling is increased when the incubation in buffer is carried out in the presence of 360-nm light. It is concluded that daughter-strand discontinuities are prevented from being rapidly sealed in the dark not because of some structural feature of the daughter-strand but because of the presence of a pyrimidine dimer on the opposite (parental) strand. "Photoreactivation-stimulated gap filling" is dependent on the polA(+) and recA(+) but not the exrA(+) genes. It is suggested that the removal of the dimer allows gap-filling by DNA polymerase I and polynucleotide ligase. The recA(+) gene may be needed at a very early stage, possibly for gap stabilization.     

The coenzyme A requirement of mammalian fatty acid synthetase: evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem.255, 1388–1392). When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl-S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C₆-C₁₆; addition of CoA promoted elongation of the acyl-S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl-N-acetylcysteamine and S-crotonyl-N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl-N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl-S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl-S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.     

Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells.

To investigate the antiviral CD4+ T cell response in coronavirus MHV-JHM-induced encephalomyelitis, spleen and thymic lymphocytes from diseased rats were stimulated in culture with virus Ag, expanded and tested for their specificity to viral proteins and nucleocapsid (N) and spike (S) proteins that had been expressed in bacteria. A strong T cell response specific for N was measurable during acute disease, whereas S-specific T cells were only detectable in rats with a later onset of disease. CD4+ T cell lines with specificity for virus and either N or S protein were established and their influence on the course of a mouse hepatitis virus-JHM infection was investigated. All lines were of the CD4+ phenotype. Both N and S protein-specific CD4+ T cells conferred protection to infected Lewis rats and reduced the amount of infectious virus in the central nervous system. After transfer of CD4+ T cells and challenge with virus, an increase in the antiviral IgM response occurred, but neutralizing antibodies were not detectable during the period of virus clearance. Previous CD8+ cell depletion did not abrogate protection mediated by CD4+ T cell line transfer.     

Polymorphisms in the umuDC region of Escherichia species.

The umuDC operon of Escherichia coli encodes mutagenic DNA repair. The umuDC regions of multiple isolates of E. coli, E. alkalescens, and E. dispar and a single stock of E. aurescens were mapped by nucleotide hybridization. umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long. Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract. Restriction site polymorphisms were not found around the DNA repair gene recA or polA. The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons. Possible mechanisms for the generation of these variants are discussed.     

Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and tri-n-butyltin chloride (Bu₃SnCl) on proline transport, proton uptake and the transmembrane pH gradient in intact cells have been compared in a CCCP-resistant mutant strain Escherichia coli UV6, and its parent strain, AN180. CCCP and Bu₃SnCl inhibited proline uptake in AN180 but the pH gradient was affected only by CCCP. Neither uncoupler affected the pH gradient in UV6 although inhibition of proline uptake occurred at high concentrations. CCCP caused efflux of accumulated proline in both strains but Bu₃SnCl was ineffective. Bu₃SnCl did not prevent the efflux of proline induced by CCCP, indicating that Bu₃SnCl had not inactivated the transport carrier. In contrast with the parent strain, CCCP failed to reverse the oxidation-dependent inhibition of the phosphotransferase system in UV6 even at concentrations causing inhibition of proline uptake. The phosphorylation potential of UV6 with succinate as substrate was lower than in AN180. This was associated with a 10-fold higher concentration of phosphate in succinate-grown UV6 than in AN180. These results suggest that CCCP and Bu₃SnCl have different sites of action on the membrane energization system of intact cells of E. coli. A possible explanation of the differences between AN180 and UV6 is that the energization system is altered in the CCCP-resistant mutant. Both uncouplers stimulated the uptake of protons by intact cells to the same extent in UV6 as in AN180. In UV6, and in AN180 with Bu₃SnCl, this was not accompanied by effects on the transmembrane pH gradient. The extent of proton uptake appeared to be related to the level of the highly anionic membrane-associated oligosaccharides in the periplasmic space. It is proposed the outer membrane acts as a partial barrier to protons and that the uncouplers can equilibrate protons between the extracellular medium and the periplasmic space in intact cells.     

Adenovirus-induced liver pathology is mediated through TNF receptors I and II but is independent of TNF or lymphotoxin.

Mice infected with an adenovirus mutant in which the E3 region is deleted, including TNF-resistance genes, develop fatal liver pathology within 3-4 days after infection. At least 10-fold more wild-type virus was needed to cause comparable pathology. These results indicate that the E3 region is critically involved in modulating the pathogenesis of adenovirus infection and that TNF may play a role in liver damage. To explore the latter possibility, the course of disease was examined in infected mice lacking TNFR-I and/or TNFRII, TNF only, or both TNF and lymphotoxin-alpha. Only mice lacking both TNFRI and TNFRII were protected from the lethal affects of the mutant adenovirus. Mice deficient in TNF or TNF and lymphotoxin-alpha displayed the fatal pathology. This outcome is consistent with the existence of another related ligand that binds TNFRI/II to mediate liver damage during infection with this mutant.