Fate of Bacteriophage   DNA After Adsorption by Haemophilus influenzae

Newman, Chris; Stuy, Johan H.

doi:10.1099/00221287-65-2-153

Cited by 9 publications

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“…influenzae differ in the ability to take up X DNA (13); modification systems specified by episomal elements might be expected to differ in different laboratory strains of the same organism. Such differences might also account for the quantitative differences among strains observed by Newman and Stuy (13) and ourselves.…”

Section: Discussionmentioning

confidence: 99%

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

1974

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Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either singleor double-strand breakage upon exposure to competent H. influenzae. The deleterious consequences attending the uptake and integration of heterologous deoxyribonucleic acid (DNA) by transformable Haemophilus influenzae were presented by Steinhart and Herriott (18); their experiments demonstrated that incorporation of H. parainfluenzae DNA into the H. influenzae chromosome was lethal for the majority of the recipient cells. Earlier experiments by Schaeffer et al. had shown that H. parainfluenzae DNA and wildtype H. influenzae DNA were equally effective in competitively inhibiting transformation by genetically marked H. influenzae DNA (15). Preparations of DNA from other genera varied considerably in their competing abilities when freshly made, but lost the ability to compete with H. influenzae DNA upon storage. No explanation of this anomalous instability was offered; this, together with the low competence of the cultures used by Schaeffer, makes the interpretation of these experiments uncertain. More recently, Newman and Stuy showed that certain cultures of H. influenzae could take up (but not integrate) X phage DNA (13). Considerable variation in the extent of uptake of X DNA was observed among different sublines of H. influenzae by these workers, who suggest that the bacteria may possess both extracellular and intracellular systems which are capable of recognizing homologous DNA. The present paper provides evidence demon-'Present address: Children's Hospital of Michigan, Detroit, Mich. 48201.

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Section: Discussionmentioning

confidence: 99%

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

1974

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“…Explanations of this phenomenon can be based on either (i) reduced uptake of the heterospecific deoxyribonucleic acid (DNA), (ii) the presence of so-called restriction enzymes (32) in the recipient that destroy the genetic integrity of adsorbed nonmodified DNA either before or after integration, or (iii) the lack of homology between donor and recipient DNA. Although competent Haemophilus influenzae Rd cells show a reduced uptake of unrelated foreign DNA (15,28), the uptake of heterologous Haemophilus parainfluenzae DNA appears to be normal (18,34). Moreover, the molecular fate of this DNA is comparable to that of homologous DNA after uptake (3,18,34,36).…”

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Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation

Stuy¹

1976

J Bacteriol

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Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a, b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterospecific transfection was at least 100 times lower than homospecific transfection in both r+ and rrecipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.-A general feature of heterospecific bacterial genetic transformation is the low efficiency of marker transfer from donor to heterospecific recipient (for a general review, see reference 1; for Haemophilus, see references 3, 16, 18, 27). Exceptions have been observed, however (14). The extent of the reduction is particular for a given marker. Explanations of this phenomenon can be based on either (i) reduced uptake of the heterospecific deoxyribonucleic acid (DNA), (ii) the presence of so-called restriction enzymes (32) in the recipient that destroy the genetic integrity of adsorbed nonmodified DNA either before or after integration, or (iii) the lack of homology between donor and recipient DNA. Although competent Haemophilus influenzae Rd cells show a reduced uptake of unrelated foreign DNA (15, 28), the uptake of heterologous Haemophilus parainfluenzae DNA appears to be normal (18,34). Moreover, the molecular fate of this DNA is comparable to that of homologous DNA after uptake (3,18,34,36). This appears to rule out a major role of restriction enzymes at the preintegration stage. It has also been shown that H. parainfluenzae DNA is integrated into the H. influenzae genome (18,34). This integration causes the death of a considerable fraction of the recipient cells through induction of the defective prophage (29) that is present in the Rd strain used most widely (4, 33). Since a presumably cured strain (2, 4) is insensitive to this lethal effect and since heterospecific transformation is also normally inefficient in this strain (29), the prophage induction phenomenon is not believed to be the explanation for the reduced heterospecific transformation efficiency. The lowered marker transfer is thus probably caused by events that follow integration (for a thorough discussion, see reference 17...

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“…The fact that breakdown may occur outside the cell argues against such a view unless DNA integration occurs at the cell surface. The greatly reduced degradation of adsorbed heterologous DNA (Newman & Stuy, 1971) suggests that it may be. We believe, however, that studies such as the ones reported here and by Newman & Stuy (1971) cannot give us a clear answer since they employ different DNAs.…”

Section: Discussionmentioning

confidence: 99%

Degradation of Adsorbed Transforming DNA by Haemophilus influenzae

Stuy

Have

1971

Journal of General Microbiology

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S U M M A R YRadioactively labelled transforming DNA is rapidly and irreversibly adsorbed by competent Haemophilus influenzae bacteria. Upon incubation, about 20 yo is degraded within 15 min. to acid-soluble products. Thereafter there is little change. Degradation products were identified by Dowex column chromatography as inorganic phosphate, nucleosides, small and large oligonucleotides. Mononucleotides were not observed. These products were always found in the medium while little, if any, was observed in the cells. It is concluded that the degradation process may take place outside the cells. It involves at least an endonuclease and a phosphatase.

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Fate of Bacteriophage DNA After Adsorption by Haemophilus influenzae

Cited by 9 publications

References 4 publications

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation

Degradation of Adsorbed Transforming DNA by Haemophilus influenzae

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