Further Studies of Infectious Dna Extracted From Mycobacteriophages

Abstract

In the previous publication (1) it was reported that deoxyribonucleic acid (DNA) extracted from mycobacteriophage D29 could infect its host, Mycobacterium smegmatis 607. No alteration in the structural integrity of the bacteria such as the production of protoplasts was required; caltures susceptible to intact phage could also be infected with phage D N A .This investigation has been extended to include three additional mycobacteriophages. I n the experiments reported here, D N A extracted from D4 and D32 but not that from D28, was found to be infectious. A t t e m p t s to extend the host range of intact phage with phage D N A were not successful. Materials and MethodsThe source and preparation of reagents, enzymes, buffers, and chromatography procedures were the same as those described in the preceding publication (1). One additional buffer used in this study, Versonate-potassium trichioroacetate (V-KTCA), Nutrional Biochemicals Company, Cleveland, Ohio, was prepared according to Weil (2). Versonate buffer was composed of 0.2 g disodium Versonate, NaCI 8.0 g, KC1 0.2 g, Na2HPO, 1.15 g, KHdaO4 0.02 g, distilled water to 1000 ml. Potassium trichioroacetate: 0.15 ~, pH 7.6. The final V-KTCA buffer was made up of 1.5 parts Versonate and 2.5 parts potassium trichioroacetate. Compounds not previously described (1), will be noted in the text.Pkages.--D28, D29, D32, and D4 were obtained originally from Dr. Seymour Froman.They were isolated by soil-enrichment techniques and except for D4, are active against certain strains of both virulent and saprophytic acid-fast bacilli; D4 is active against Mycobacterium smegmatis 607 (3, 4). Methods for the production of high-titered lysates and for phage assay have been described (5).Baaeria.--Mycobaaerium smegmatis ATCC 607 (propagating host for D4 and for D29), Mycobacterium sp. ATCC9033 (propagating host for D32), and Mycobacterium sp. ATCC 9626 (propagating host for D28) were employed. These bacteria will be referred to in the text by their ATCC numbers. Two additional species of Mycobacterium were employed in hostrange experiments. They were obtained from Dr. Froman and, according to him, one is M.phlei (F 89) and the other M. smegmalis (F 87). The latter can be distinguished from 607 by its phage susceptibility pattern. These bacteria will be referred to in the text as F 89 and as F 87. Stock cultures were maintained, and log-phase cultures prepared as described (5).

Biochemical and morphological characterization of mycobacteriophage R1

“…denaturation studies yielded G+C contents of 66 and 65%, respectively, for Rl DNA. These values are consistent with those found for other mycobacteriophages (27). Furthermore, the sharp transition curve that we observed during the thermal denaturation studies is characteristic of double-stranded DNA (14).…”

Recombination between Mycobacterium smegmatis Strains Jucho and Lacticola

“…The cells grown in liquid or on solid media were tested for the presence of prototrophic cells. In all the systems, no significant increase of prototrophic colonies was observed comparing with DNA extracted from mycobacteriophages [15,20] and adsorbed cellular DNA [13] suggested strongly the former possibility although no report on successful transformation has yet been published [14,17,19]. This possibility, however, is almost excluded by the following experimental results: 1) When DNA extracted from one strain was employed instead of the bacterial cells, no recombinant was produced;…”

Cytosine-phosphate-guanine (CpG) motifs are sensitizing agents for lipopolysaccharide in toxic shock model

“…Bacterial DNA differs from eukaryotic DNA by the presence of sequences containing unmethylated cytosinephosphate-guanine (CpG) motifs conferring immune stimulatory activity that is not seen with eukaryotic DNA [1,2,3,4,5,6,7,8,9,10,11]. It is therefore postulated that immune activation by unmethylated CpG motifs may be part of the innate immune defense mechanisms triggered by structural patterns present in microbes [8,12].…”