Metabolic and nutritional factors influencing the development of competence for transfection of Bacillus subtilis

Despite the remarkable diversity among the bacterial species which can be transformed by purified deoxyribonucleic acid (DNA), there appears to be considerable uniformity in the mechanism of the transformation process. The steps of the transformation process have been known for some time and have been considered in several reviews (13, 15, 19), but for clarity the common features are briefly summarized below. (i) A reversible association of the DNA with recipient cells occurs. (ii) Irreversible binding and nonspecific incorporation of high molecular weight DNA, achieved by means of an energy-consuming process, result in a state of deoxyribonuclease insensitivity. The recipient cells have a unique and transient physiological capability, referred to as competence, which enables them to carry out this process. This capacity is itself controlled by at least one genetic locus. (iii) A period of latency follows during which nonhomologous DNA (and much homologous DNA) is degraded to acid soluble components. (iv) Single-stranded portions of donor molecules which have survived degradation are integrated to replace approximately homologous sections of recipient DNA. This process resembles repair synthesis of DNA and not extensive DNA synthesis, since the newly integrated region is probably not much greater than several thousand nucleotides in extent (2). Under normal physiological conditions, the competent cells cannotincorporate single-stranded DNA directly; however, by manipulating the recipient culture during certain stages of growth, or stressing the physical boundaries of the cell, various species can be induced to take up DNA under conditions where it would otherwise be excluded (4, 5, 7, 12). Finally, (v) segregation and expression of the newly incorporated genetic information occurs, leading to the production of genetically stable progeny bacterial cells that are phenotypically distinguishable from the original recipient population. Special note should be made of the fact that, under the normal physiological conditions which are required for competence development, none of the transformable species has been shown to be capable of conjugation. This leads to the speculation that transformation may represent the normal means of genetic exchange in these species. Spizizen and co-workers made a very significant contribution to the study of microbial genetics when they identified and characterized the transformation system in Bacillus subtilis (1, 24, 25). This system has numerous advantages over other transformation systems. Important among these are the ability of B. subtilis to grow and develop competence in a simply defined glucose-salts growth medium, its nonpathogenicity, and its well-characterized built-in system of cellular differentiation-the process of endospore formation. However, the relative inefficiency of the B. subtilis system is one serious disadvantage for many genetic studies. In the system which Spizizen characterized, only 1 to 4% of the recipient population is actually in a state of competence when this characteristic is being maximally expressed. This must be compared with levels of competence which exceed 90% in other species. The following considerations led us to undertake a study of the physiology of competence development in B. subtilis. (i) The expression of competence is a peculiar prerequisite for DNA uptake in all transformable systems known, yet there appears to be little or no uniformity in the characteristics of the formation of this stage among the widely diverse transformable species. A better understanding of the physiology of the cells involved might bring to light some unifying characteristics. (ii) In recent years, considerable attention has been focused on the functioning of B. subtilis genetic material as it relates to the