Genetic transformation in Haemophilus parainfluenzae clinical isolates.

Haemophilus parainfluenzae isolates recovered from patients with respiratory diseases were studied for their ability to undergo genetic transformation by isogenic DNA. Two chromosomal markers, streptomycin resistance and nalidixic acid resistance, were tested for transformation efficiencies in H. parainfluenzae recipients from three biotypes. Most efficient in transformation was biotype II, followed by biotype I, while biotype III was nontransformable. Lack of transformation was not owing to poor donor activity of DNA, but to inability of the cells to develop competence. Strains that formed clumps in liquid media were nontransformable. Since the transformable biotype II is one of the prevalent biotypes world wide, one can speculate that DNA transformation probably plays a major role in the spread of drug resistance in H. parainfluenzae.

Growth characteristics of V factor-independent transformants of Haemophilus influenzae.

Haemophilus influenzae is a V factor-dependent species. A plasmid conferring V factor independence in Haemophilus parainfluenzae and Haemophilus ducreyi was transferred to plasmid-free H. influenzae Rd by DNA transformation. The growth characteristics of the transformants in a complex and a chemically defined medium were compared, and the ability of several exogenous pyridine nucleotides and precursors to support growth was examined. Although the transformants appeared to be V factor independent in a complex medium, in a chemically defined medium they exhibited both V factor-dependent and nicotinamide-dependent growth. Because of the inability of the plasmid-free H. influenzae Rd to utilize nicotinamide for growth, it was concluded that the genes conferring this function were plasmid linked. Our results indicate that the V factor requirement, as it is presently defined, is not suitable to serve as a definitive taxonomic criterion for species determination in the family Pasteurellaceae.

Plasmid-mediated NAD independence in Haemophilus parainfluenzae.

The location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5.25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0.1 to 0.2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid 'curing' effect with a frequency of 'curing' of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5.25 kb plasmid.

Naturally occurring NAD-independent Haemophilus parainfluenzae.

Four, NAD-independent, clinical isolates of Haemophilus parainfluenzae were recovered from a genital ulcer, a purulent skin lesion, a sputum specimen and a throat swab respectively. With the exception of NAD requirement, the strains exhibited the biochemical characteristics of H. parainfluenzae biotype II. The genetic relationship between these isolates and a standard strain of H. parainfluenzae was determined by testing transforming activities of two chromosomal markers, streptomycin resistance and nalidixic acid resistance. The clinical isolates were efficient donors and recipients in transformation. In addition, we demonstrated transfer of the genes conferring NAD independence to typical, NAD-requiring H. parainfluenzae and Haemophilus influenzae strains.

Genetic transformation in encapsulated clinical isolates of Haemophilus influenzae type b.

Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x 10(-4) to 1 x 10(-6). The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.

Uptake of plasmid deoxyribonucleic acid by Haemophilus.

The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.

Occurrence of pili on and adhesive properties of Haemophilus parainfluenzae.

Of 20 clinical isolates of Haemophilus parainfluenzae, 13 produced a mannose-resistant hemagglutinin that agglutinated erythrocytes from chickens, horses, rabbits, and sheep. Examination with the electron microscope showed that only strain HR-885 was pilate. Grown in static liquid culture, the 12 hemagglutinating, nonpilate isolates formed small, tightly packed clumps, whereas strain HR-885 formed large, loosely packed clumps. However, seven isolates did not produce a hemagglutinin, did not clump, and were nonpilate.

Biological properties of a Haemophilus influenzae restriction enzyme, Hind I.

A type I restriction enzyme from Haemophilus influenzae, Hind I, which requires adenosine 5' -triphosphate and 5-adenosyl methionine, was studied for its activity on transfecting and transforming deoxyribonculeic acid (DNA). The enzyme reduced the size of unmodified bacteriophage S2 DNA from 37 X 10(6) daltons to approximately 10 X 10(6) daltons, but did not affect modified S2 DNA. Unmodified transforming DNA was attacked in vitro by Hind I; however, relatively low levels of inactivation were obtained for single markers, and linked transformants were inactivated as a function of the distance between markers. In contrast, unmodified bacterial DNA was not inactivated in vivo for either single or linked markers by the Hind I restriction system, probably because the segments generated by Hind I were still capable of being integrated in vivo. The lack of preferential inactivation of markers by the enzyme suggests that it makes random breaks in the DNA.

Restriction and modification of bacteriophage S2 in Haemophilus influenzae.

The major conclusion from these studies is that variants of Haemophilus influenzae Rd which restrict and modify phage S2 are metastable and capable of giving rise to one another with high frequency. Nonrestrictive RdS cells segregate spontaneously to the restricting, modifying phenotype in about 5% of the progeny of a single clone. The restrictive cells derived from RdS revert to the nonrestrictive phenotype in 15 to 25% of the progeny of a single clone. These frequencies are not appreciably affected by treatment with acriflavine or ethidium bromide, compounds which affect plasmid stability, or by nitrosoguanidine, a powerful mutagen. The genetic locus for restriction and modification of bacteriophage S2 is found to have a chromosomal position between the biotin and proline loci. Restriction-modification of phage S2 has been shown to be a function of its deoxyribonucleic acid (DNA) in that transfection with S2 phage DNA or prophage DNA is subject to host restriction and modification. An enzyme preparation, which contains endodeoxyribonuclease but no appreciable exonuclease activity, from mutant H. influenzae com(-10) did not restrict phage S2.RdS DNA or prophage DNA transfecting activity, indicating that this endodeoxyribonuclease is not responsible for phage restriction. A new restriction enzyme isolated from H. influenzae Rd was found to be the major enzyme involved in the restriction of bacteriophage S2. The enzyme inactivated the transfecting activity of unmodified phage DNA but did not attack modified phage DNA. Unlike endodeoxyribonuclease R, this enzyme requires adenosine triphosphate and S-adenosylmethionine.

Separation of specific segments of transforming DNA after treatment with endodeoxyribonuclease.

Hemophilus parainfluenzae endodeoxyribonuclease was used to degrade the DNA of H. influenzae and to follow the biological activity of 14 markers associated with this DNA. It was found that some H. influenzae markers were completely inactivated by endodeoxyribonuclease treatment, while others appeared to retain all or almost all of their original activity. The bulk of the H. influenzae DNA was reduced to double-stranded pieces of the order of 8 x 10(5) to 1 x 10(6) daltons. Velocity sedimentation of the DNA in sucrose gradients disclosed that markers that retained biological activity were present in DNA particles that were of the order of 1 x 10(6) daltons or larger, and indicated a close correlation between the size of the DNA fragment and the amount of biological activity retained. These data suggest that H. parainfluenzae endodeoxyribonuclease breaks DNA at specific sites. The nal(r) marker was shown to have twice as much biological activity after treatment with endodeoxyribonuclease when assayed at saturating DNA concentrations. In the linear portion of the DNA dose-response curve, the biological activity of this marker was reduced 3- to 10-fold compared to untreated DNA (in accord with the reduced size of its DNA). These data demonstrate a specific enrichment of the nal(r) marker by about 6- to 20-fold, and suggest a technique for the separation and purification of specific segments of DNA.

Action of haemophilus endodeoxyribonuclease on biologically active deoxyribonucleic acid.

An enzyme similar to that described by Smith and Wilcox (15) for Haemophilus influenzae which attacks foreign deoxyribonucleic acid (DNA) but not its own has been isolated and purified from H. parainfluenzae. The enzyme degrades foreign DNA to limited sizes and can destroy the transforming activity of H. influenzae and Bacillus subtilis DNA. The enzyme can also destroy the biological activity of H. influenzae phage and prophage DNA. On the other hand, the H. influenzae endodeoxyribonuclease can destroy the transforming activity of H. parainfluenzae DNA but not its own DNA. It also attacks B. subtilis DNA and its transforming activity.

Induction of colicin 1a at high temperature.

Induction of colicin la at a high temperature (42 C) was demonstrated in a wild strain of Shigella sonnei. After transfer of the Col factor, a similar effect was observed in those recipient cells acquiring colicinogeny. The induction is suggested to be due to the presence of a thermosensitive colicin repressor. The wild-type strain segregated sensitive cells and showed heterogeneity in colicin production.

T-related bacteriophages isolated from Shigella sonnei.

The properties of three T-related phages-35, 55, and 3201-isolated from Shigella sonnei were studied. They were similar with respect to morphology of plaques, duration of the latent periods, lysis inhibition effect, and serological characteristics. These phages closely resembled the T-even phages. Phages 3201, 35, and 55 had the same host range and receptor specificity as phage T2.