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.

Gyrase mutants affect mutation in a localized region of Haemophilus influenzae.

Spontaneous mutation was greatly increased in a localized region of the chromosome of Haemophilus influenzae, but not at other loci, by a nov gene mutation called novC that increased DNA supercoiling. Another nov gene mutation, called novD, decreased spontaneous mutation in the same localized region and depressed DNA supercoiling. Both mutations, which code for the gyrase B subunit, have been cloned, and the cloned versions also altered mutagenesis and supercoiling in a similar fashion as the two mutations on the chromosome, although novC on the plasmid caused somewhat less mutation than on the chromosome. We postulate that the effects of the gyrase B mutations on the chromosome result from their effects on supercoiling because of increased gyrase near its site of production. The fact that the novC on a plasmid does not cause mutagenesis except in the same localized region that is altered by this mutation on the chromosome, is difficult to explain. One possibility is that there is a complex of proteins in this region which is necessary for the effects on supercoiling and thus, also on mutagenesis.

[Lack of foto reactivation of the Haemophilus influenzae transforming DNA mutated with near ultraviolet light (325-400nm)]. / Ausencia de fotorreactivación en el DNA transformante de Haemophilus influenzae, mutado con luz de ultravioleta cercano (325-400 nm).

Three extracts from Saccharomyces cerevisiae were obtained by salting out with ammonium sulfate, these were I-G, EFRL-II-G and III-G. Fraction EFRL-II-G showed the highest photoreactivating activity on DNA str2000 irradiated with far UV light. However, the same fraction did not reactivate DNA str2000 previously inactivated by near UV irradiation. We think that the inactivation by near-UV was not due to photochemically-formed pyrimidine dimers. Decrease in spontaneous mutation frequency of cells transformed with DNA str2000 irradiated with near-UV light, was the same with the DNA treated with active or heat inactivated EFRL-II-G, therefore we may conclude that DNA lesions responsible for the effect are difference to pyrimidine dimers.

[Decrease of spontaneous mutations in Haemophilus influenzae caused by transformation with its own DNA irradiated with near-ultraviolet light]. / Disminución de la mutación espontánea de Haemophilus influenzae por transformación con su DNA irradiado con luz del ultravioleta cercano.

Transforming DNA containing the streptomycin resistance marker, was irradiated for 8 h with broad near ultraviolet light (325-400 nm) at pH 4.8, and the inactivation kinetics determined. After selection of streptomycin resistant transformants, they were grown until a turbidity of 150-200 Klett units. In these cultures we looked for new markers coming from the irradiated transforming DNA. We looked and found the novobiocin resistance marker and one that conveys to protoporphyrin IX utilization, measured as an increase in the mutation frequency of these markers in the streptomycin resistant population. In other experiments, we found a decline in spontaneous mutation frequency for the same markers in the cells transformed with irradiated DNA. This last finding rises the possibility of alterations on the mutator genes as a result of near ultraviolet irradiation.

In vitro mutation of Haemophilus influenzae transforming deoxyribonucleic acid by ultraviolet radiation at -70 degrees C.

Previous studies have shown the non-mutability of Haemophilus influenzae either by UV irradiation of the cells or by irradiating the transforming DNA and transformation of competent cells. In the present work, we present evidence of transforming DNA mutation in vitro by UV irradiation at -70 degrees C, which upon transformation of competent cells showed a rise in the mutation frequencies of novobiocin resistance of the order of several hundredfold. Also we performed experiments using the UV-irradiated DNA either sonicated or DNase-treated, which allowed us to propose that such rise in mutation frequency is probably due to the integration of DNA carrying premutagenic photoproducts to the recipient cells' genome. We think that the key point was the low temperature at which the DNA was irradiated in order to obtain the mutagenic effects, since it is likely that at -70 degrees C, the main photoproducts are not the cyclobutane dimers, but are the spore photoproducts, which are probably responsible for the damage that leads to mutagenic effects.

Lethal and mutagenic action of hydrogen peroxide on Haemophilus influenzae.

The lethal and mutagenic effects of H2O2 on wild-type Haemophilus influenzae Rd and on uvr1, uvr2, rec1, and rec2 mutant strains were studied. The first two mutants are sensitive to UV, and the second two are defective in recombination. Rd, urv1, and rec1 strains were more sensitive to the killing effect of H2O2 treatment than were uvr2 and rec2 strains. There were peaks of mutagenesis at two H2O2 concentrations over a range of 30 to 275 mM. Our results suggest a specific repair of H2O2 damage that is independent of the Uvr2 and Rec2 gene products. Sensitivity to the killing effect of H2O2 and to the lethal action of near-UV light were similar for Rd and uvr1 strains. This finding suggests that the mechanisms of killing by and repair of H2O2 damage may have some overlap with those of near-UV radiation.

Mutagenic and lethal action of polychromatic near-ultraviolet (325-400 nm) on Haemophilus influenzae in the presence of nitrogen.

The lethal effect of polychromatic near-UV light (325-400 nm) on Haemophilus influenzae was 8 times higher under aerobic than anaerobic irradiation. This light increased the frequency of mutation to novobiocin resistance and ability to utilize protoporphyrin IX. The slope of mutagenic effect at low doses appeared greater for the aerobic than for the anaerobic group. We concluded that polychromatic near-UV mutation of H. influenzae under anaerobic irradiation was caused by direct oxygen-independent action on DNA.

Acción sobre haemophilus influenzae de su ácido desoxirribonucléico transformante irradiado in vitro con luz ultravioleta a -70- / Action on haemophilus influenzae of its transforming deoxyribonucleic acid irradited in vitro with ultraviolet light at -70-C

La irradiación con luz ultravioleta a - 70-C de DNA transformante desnaturalizado, el cual fue posteriormente sonicado y renaturalizado con DNA no irradiado, así como el DNA no irradiado y sometido a los otros tratamientos, provocaron un fuerte efecto letal sobre H. influenzae, el cual fue detecto al efectuarse la cuenta viable de las mezclas de transformación, encontrándose la disminución de la viabilidad de las células de un 98% para el caso del DNA irradiado y de un 97.2% para el DNA sin irradiar. Pensamos que la letalidad pueda deberse, tanto a la integración al genóforo de la célula receptora de los marcadores letales provados por la luz UV, como a la activación de un fago defectuoso causada por la penetración del DNA transformante irradiado o no con luz UV. La irradiación con luz UV del DNA transformante desnaturalizado, el cual fue renaturalizado consifo mismo, hizo aumentar ligeramente el número de mutantes resistentes a Kanamicina en células competentes de H. influenzae. Al someterse a lisis sónica del DNA irradiado, antes de renaturalizarse con DNA no irradiado, el resultaod obtenido fue aproximadamente el mismo que en el caso anterior. Sin embargo, cuando el número de mutantes resistentes a novobiocina se corrigió por la cuenta viable de la mezcla de transformación y el efecto mutagenético se cuantificó con relación a las frecuencias de mutación, encontramos un aumento de dichas frecuencias de 74 veces respecto a los testigos de células no tratadas con DNA. Este efecto puede, en principio, deberse a la integración en el genoma receptor de las lesiones mutagenéticas causadas por la irradiación con luz UV a - 70-C del DNA transformante, mas la afirmación concluyente de esto, requerirá la demostración de que dicha integración se lleva a cabo. Proponemos que estas lesiones mutagenéticas pudieran ser del tipo 5-timinil-5, 6-dihidrotimina. Fue interesante que del SNA no irradiado con luz UV, provocara también un aumento aunque menor al producido por el DNA irradiado, de las frecuencias de mutación. La explicación de este hecho, necesitará de un análisis experimental detallado posterior, utilizando mutantes rec y uvr de H. influenzae

Acción del calor sobre haemophilus influenzae / Effect of heat in Haemophilus influenzae

Las temperaturas de 45, 47, 50 y 55-C mostraron un efecto letal sobre H. influenzae, siendo más acentuado cuando se calentó en un medio sintético que en extracto cerebro-corazón. A 45 y 47-C fue muy notable un "hombro" de resistencia a tiempos cortos de calentamiento. Se observaron dos pendientes de letalidad, lo cual sugiere dos mecanismos de inactivación por el calor y la presencia de procesos reparativos en la célula. La incubación a 37-C en extracto cerebro corazón de células previamente inactivadas a 45 ó 47-C produjo una reversión del efecto letal. La curva de inactivación a 45-C mostró tres zonas el "hombro" inicial de resistencia, en seguida la fase exponencial de inactivación y finalmente otra zona de resistencia entre los 10 y 30 minutos de tratamiento. Esta última zona sugiere la presencia de una mutante termoresistente. Todos los datos anteriores sugieren al ácido desoxirribonucleico como uno de los blancos importantes al tratar a Haemophilus influenzae con el calor

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.

Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae.

Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juárez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell.

Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae.

The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNov1. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. Our hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.