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The acetone-soluble fraction extracted from lyophilized cells of Mycobacterium smegmatis ATCC 607 inhibited D4, a species-specific phage active against M. smegmatis. Evidence is presented indicating that the D4 inhibition was caused by the phage receptor substance(s) contained in this fraction. A fraction eluted from silicic acid with chloroform-methanol (95:5, v/v) showed the strongest inhibition of D4 phage. This fraction contained sugars and amino acids, and its infrared absorption spectrum was practically identical with those of the mycoside C isolated from the other species of mycobacteria. Further fractionation revealed that the active material was a mixture of several closely related peptidoglycolipids all of which showed, more or less, the phage inhibition. One of the compounds was purified and partially characterized; it contains three different amino acids, allo-threonine, alanine, and phenylalanine, at a molar ratio of 1:1:1, and also three different deoxyhexoses, probably 6-deoxytalose, 3,4-di-o-methylrhamnose, and 2,3,4-tri-o-methylrhamnose. A tentative name of "mycoside C(sm)" is proposed for this substance which possesses a slightly different structure from the known types of mycoside C and is probably specific for the species of M. smegmatis. A fraction extracted from the D4-resistant mutant of M. smegmatis ATCC 607 by acetone and then by chloroform-methanol (95:5, v/v) showed no phage inhibition and had no sugar component. In addition, this fraction contained lysine, serine, and a small amount of both glycine and an unidentified amino acid.

Results of the previous investigation in which it was found that DNA extracted from D29 mycobacteriophage was infectious for Mycobacterium smegmatis 607, have been extended. DNA extracted from mycobacteriophage D4 and D32 produced plaques when plated on their respective hosts; D28 DNA, extracted in the same manner and tested under similar conditions, failed to show infectivity. Species barriers were not crossed by mycobacteriophage DNA; bacteria resistant to intact phage were not infected with the phage DNA. The efficiency of plating of the DNA is very much lower than that of intact phage; infection of a given host was not accomplished by DNA when titration for plaque formation by the intact phage was less than 10(9) PFU. The base composition of DNA extracted from the four mycobacteriophages and the three propagating hosts was very similar. The bases were paired, adenine with thymine and guanine with cytosine. A relatively higher per cent of guanine-cytosine than of adenine-thymine, was found. The buoyant density of each DNA in CsCl was linearly related to its guanine-cytosine content whereas with the exception of D28 DNA, thermal denaturation temperatures failed to show this relationship. However, the thermal transition profiles were characteristic of double stranded DNA. Additional evidence that D29 DNA forms complexes with basic proteins was obtained. Binding between calf thymus histone and between RNAase and D29 DNA readily occurs with a resultant loss in DNA infectivity. Trypsin and D29 DNA are only weakly reactive.

DNA extracted from D29 mycobacteriophage produced plaques when plated on Mycobacterium smegmatis 607. The host bacterium did not require alternation such as conversion to protoplasts; cells susceptible to infection with intact phage were susceptible to DNA. The bases found in calf thymus DNA constituted the bases of D29 DNA, adenine being paired with thymine and guanine with cytosine. The dissymmetry ratio (A + T/G + C) was 0.56, and the buoyant density in CsCl was 1.722 with a GC content of 63.77 per cent. The efficiency of plating of the DNA is very much lower than that of intact D29, and it penetrates the host at a slower rate. As does intact phage, D29 DNA requires calcium ions for productive infection of 607. D29 DNA is significantly inactivated by incubation with RNAase, but the inactivation probably results from a complexing with the DNA rather than from enzyme hydrolysis.