AN INVESTIGATION OF ENUMERATION AND DNA PARTITIONING IN BACILLUS-SUBTILIS L-FORM BACTERIA

Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-w alled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units ( cfu)). The DNA hybridization method was shown to be a reliable and rep roducible method for estimating genome numbers. Comparison of differen t L-form populations showed that the two methods of enumeration gave d ifferent values, with the deduced genome numbers much higher (by sever al orders of magnitude) than cell numbers deduced from viable cell cou nts. In contrast, when a culture of the cell-walled form was enumerate d, the discrepancy between the two methods was low (by a factor of abo ut 6) The combination of a high number of L-form genomes detected by D NA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replicatio n and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamyci n resistance. The results are discussed with particular reference to c ell division in cell wall-deficient, stable L-form bacteria.