Jane K. Setlow

Heterospecific transformation in the genus Haemophilus

SummaryThe relationship between nine Haemophilus species and Haemophilus influenzae was studied by DNA-DNA hybridization, by transformation of H. influenzae to streptomycin resistance with heterospecific DNA, by competition of heterospecific DNA for transformation by homospecific DNA and by the lethal effect of heterospecific DNA on competent H. influenzae. H. parainfluenzae, H. parasuis, and H. aegyptius DNA transformed at more than 10% efficiency when compared to homologous transformation, but only H. aegyptius demonstrated, by hybridization, a relative binding ratio of more than 80%. H. aphrophilus and H. paraphrophilus DNA demonstrated a relative binding ratio of less than 30% and transformed H. influenzae at only 10-5 the efficiency of homologous DNA, but they competed for H. influenzae transformation as well as or better than homospecific DNA. The data indicated that in some of the species sharing the common ecological habitat of the mammalian respiratory tract, sequences necessary for competition and efficient uptake into H. influenzae are present in large numbers in their DNAs, which nevertheless have little overall homology with H. influenzae DNA.

Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.

Relatedness within the family Pasteurellaceae as determined by genetic transformation

Genetic transformation studies were used to determine relatedness within the family Pasteurellaceae. Among strains with < 60% relatedness to Haemophilus influenzae based on deoxyribonucleic acid hybridization, two groups were identified; one, showing competition for homospecific transformation with H. influenzae, contained Haemophilus parainfluenzae, Haemophilus parasuis, Haemophilus aphrophilus, Haemophilus paraphrophilus, Pasteurella pneumotropica, Pasteurella multocida, and Actinobacillus actinomycetemcomitans, and the other, showing little or no competition for homospecific transformation with H. influenzae, contained Haemophilus ducreyi, Haemophilus parahaemolyticus, Actinobacillus lignieresii, Actinobacillus equuli, Actinobacillus pleuropneumoniae, and Pasteurella ureae. Such groupings support existing studies which have used only deoxyribonucleic acid hybridization or numerical analysis.

Action spectrum for lethality of near-UV light on Haemophilus influenzae and lack of mutation

Mutation and inactivation of H. influenzae have been measured following irradiation at various near-UV wavelengths. Inactivation takes place most readily at 334 nm (but is unaffected by absence of excision or postreplication repair), and decreases markedly at longer wavelengths. No induced mutations to resistance to novobiocin or streptomycin or to ability to utilize protoporphyrin instead of hemin were detected at any of the wavelengths used. There were also no detectable induced mutations in an excision-defective strain after 334-nm irradiation. These results are in contrast to the in vitro mutation of purified transforming DNA we previously observed.

DIFFERENT CHROMATOGRAPHIC BEHAVIOR OF A THYMINE-CONTAINING NEW ULTRAVIOLET PHOTOPRODUCT AND SPORE PHOTOPRODUCT

Abstract— One‐ and two‐dimensional cochromatography in five different solvents of a near UV photo‐product induced by 365 nm irradiation of DNA in aqueous solution and the ‘spore photoproduct’, 5‐thyminyl‐5,6‐dihydrothymine, produced by 254 nm irradiation of dry DNA, indicate that the two photoproducts seem to have related structures but are different.

Near-ultraviolet mutation of transforming DNA irradiated in vivo

Our previous work has demonstrated that whereas near-UV radiation is not a mutagen for Haemophilus influenzae cells, it does induce mutations in purified transforming DNA. In order to test various hypotheses concerning this difference, we have irradiated cells at 334 and 365 nm, then lysed them and assayed the DNA for induced mutations and for inactivation of transforming ability. The inactivation was only a little lower than observed with highly purified transforming DNA. The DNA irradiated in vivo was mutated at both wavelengths, but with considerably lower efficiency than was purified DNA. Neither incubation of the cells after irradiation and before lysis nor freezing and thawing the cells significantly changed the amount of mutation. It is concluded that there is some protection of the DNA against premutational lesions by the in vivo environment, but that it is not enough to account for the total lack of mutation of the cells. A probable explanation of this lack of cell mutation is that lethal lesions in the cells are induced much more readily than premutational lesions.

Repair and action spectrum of oxygen-independent lethality of near UV light on Haemophilus influenzae and lack of mutation

Haemophilus influenzae has been inactivated anaerobically at 313, 334, 365 and 405 nm, and exhibits the greatest sensitivity at 334 nm. The rec1 and uvr1 mutants show the greatest increase in sensitivity over the wild-type at 313 nm, but differences could be seen also at the other wavelengths. Anaerobic irradiation is less effective for killing at all the wavelengths than irradiation under aerobic conditions, but the greatest difference was observed at 365 nm. No induced mutation was seen as a result of anaerobic irradiation at 334 nm, although purified transforming DNA can be mutated at this wavelength.

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents.

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.

Evidence for gene silencing in Haemophilus influenzae

Evidence for gene silencing of Haemophilus influenzae involved a beta-subunit of RNA polymerase. The gene presumed silenced was rifampin resistance. The evidence that it was silencing, rather than dominance of a rifampin-sensitive marker, was that it took place when the rifampin resistance marker was on both a plasmid and the chromosome, without the presence of a rifampin-sensitive marker, as judged by lack of transformation of a rifampin-resistant cell to rifampin sensitivity by the plasmid. In addition, three compounds that are known to decrease gene silencing in eukaryotes (trichostatin A, sodium butyrate and 5-azacytidine) also decreased the presumed silencing in H. influenzae. Silencing of rifampin-resistant Escherichia coli did not take place with the plasmid from H. influenzae.

MucA protein affects spontaneous mutation in a localized region of Haemophilus influenzae.

A plasmid called pMucA, from a piece of the plasmid pKM101 (Mol. Gen. Genet 167 (1979) 317) cloned in the vector pDM2 (J. Bacteriol. 151 (1982) 1605), caused higher mutation in a local region of Haemophilus influenzae and caused even more mutation there in a strain also containing novC, the latter causing an increase in supercoiling (J. Bacteriol 164 (1985) 525). The novD mutation depressed supercoiling, and also depressed the mutation by pMucA in the local region of the chromosome. Thus, it is clear that supercoiling is an important phenomenon in spontaneous mutation of H. influenzae. The pMucA plasmid caused a number of other phenomena in H. influenzae, induced UV mutation (Proc. Natl. Acad. Sci. USA 82 (1985) 7753), decreased UV sensitivity of transforming DNA, but not cells, and UV-induced recombination of mutants of phage HP1c1. The effect of the MucA protein in mutagenesis of H. influenzae we consider to be due to the introduction of some of the E. coli functions from pKM101. We postulate that the localized mutation caused by the MucA plasmid also involved localization of the plasmid or its coded protein in the same area, resulting from binding to a homologous gene, probably rec-1, very close to the localized region.