The cell wall of Bacillus subtilis 168 is composed of two heteropolymers, teichoic acid and peptidoglycan.1 Teichoic acid is a polyglycerol phosphate with D-alanine or D-glucose on glycerol carbons 1 or 2 and a poly N-acyl hexosamine on the terminal phosphate.2-4 This polymer is linked via a peptide bond from muramic acid on the poly hexosamine to a muramic acid residue of the peptidoglycan. A phage-resistant mutant, Bacillus subtilis 168/29, isolated by Dr. B. Reilly,5 provided an opportunity to examine the alterations of the cell wall associated with phage resistance. This strain did not adsorb phages 425, 429, and SP10.5 Subsequent experiments have demonstrated that resistance to five groups of phage is acquired by a single modification of teichoic acid. This communication describes the structural alterations and the enzymatic defects in phage-resistant strains of B. subtilis. Materials and Methods.-Bacterial and phage strains: Bacillus subtilis, strains 168, 168/29,6 and H, later reclassified as Bacillus amyloliquefaciens,7 were used. Mutants of B. subtilis 168 resistant to phage were obtained spontaneously and by treatment of the parental strain with ultraviolet light8 or with nitrosoguanidine.9 Clones were selected which failed to adsorb phage and did not liberate phage after repeated subculture. A mutant of B. subtilis 168 resistant to SP02, Mu8u5u5 (SPO)/SPO2 here designated as 70, was provided by Dr. Loubelle Boyce. Indicator strains were: B. subtilis 168 for phages c/A, 025, SPO2, and SP3; B. subtilis W23 for SP10, and B. amyloliquefaciens H for 429.Phages 01, 025, ,29,10 and SP1011 were kindly supplied by Drs. B. Reilly and J. Spizizen and SPO2 by Drs. LB. Boyce and R. Romig. Lysates were prepared according to Reilly6 and radioactive phage by growth in a tryptophan-thymidine double auxotroph of B. subtilis 16812 using minimal minus phosphate medium at pH 713 containing 10 ,ug per ml thymidine and either 2.5 ,uc H3 thymidine or 10 Mc p32 per ml. The phage were harvested as recommended by Reilly and Spizizen,6 dialyzed for 25 hr against 3 changes of 100 vol of PM buffer at 40C, filtered through a Millipore filter, and stored at 40C.Phage assay: Cell walls, prepared from 5-hr cultures of B. subtilis,4 were incubated with phage at either 37°C or 4°C for 5, 15, or 30 min in PM or PAB. The walls were removed by centrifugation at 10 Krpm for 10 min at 4°C, and the supernatant solution was assayed for phage by plating an appropriate dilution in semisolid agar over TBAB; for SP02, Ivanovic's semisolid agar"4 was used over AK agar (Difco).Electron microscopy: The phage and phage-wall complexes were stained with phosphotungstic acid"5 and examined in a Hitachi model 11 electron microscope.Extract and membrane preparation: Wild-type B. subtilis strain 168, and the phage-resistant mutants, were grown overnight in pH 7.2 tryptose broth (gm/liter: Bacto-tryptose, 10; Bactobeef extract, 3; and NaCl, 5). Minimal medium4 was inoculated to optical density of 10-15 Klett units (filter no. 66), and incubated 4 hr at 370C ...

Lysates of the virulent bacteriophage SPP1 were shown to be capable of mediating generalized transduction. Suppressible mutants of this bacteriophage (sus) were capable of transduction at a lower multiplicity of infection than virulent SPP1. Linkage analysis demonstrated that bacteriophage SPP1 transduced segments of the genome equal in size to that transferred by SP10. This bacteriophage should be useful in analyzing the regions of the genome where PBS1 appears to give anomalous results.

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Antibody to DNA in human serums can be detected by the ammonium sulfate method. This sensitive and specific technique, which measures the primary interaction between DNA and antibody to DNA, is based on the observation that free DNA is soluble in 50-percent saturated ammonium sulfate whereas antibody-bound DNA is insoluble.