The composition of a defined nongrowth medium used in stage II development of competence of Haemophilus influenzae affects the course of this development. The development of competence in two nongrowth media, M-IV and M-V, is rapid, logarithmic, and independent of the cell concentration. This last property indicates that there is probably no transfer of a competence factor from competent to noncompetent cells, in contrast to results reported for other organisms. Levels of competence reached in these completely defined media are such that 1 to 5 % of the cells are transformed in the presence of an excess of marked deoxyribonucleic acid. The method of evaluating competence, which depends on the frequency of multiple independent transformations, has been reexamined. This and other methods are compared on samples taken from a culture during development of competence.

A B S T R A C T A procedure has been developed for obtainingHem0philus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent ceils.

/mSTRAC¢H~mopkilus influen~--transforming DNA, which has been inactivated by ultraviolet radiation, is reactivated by visible light in the presence of a cell-free extract of Eschcric~ia coli B.The time rate of reactivation is increased by increasing the E. coli extract concentration, the temperature, and the intensity of illumination.Only DNA containing an ultraviolet-damaged genetic marker exhibits increased transforming activity after treatment with the photoreactivatlng system.The reactivating capacity of the extract remains in the top supernatant after centrifugation at 110,000 X g for 1 hour and is not present in the pellet. This capacity is destroyed by heating to 90°C. for 1 minute.The active system of the E. cdi extract is separable into dialyzable, heat-stable and non-dialyzable, heat-labile fractions. The dialyzable fraction contains at least one component which limits the max-imum degree of recovery attained.Photoreactivation, the reversal of short wave length ultraviolet effects on organisms by subsequent treatment with visible light, was first specifically described by Keiner (1, 2) for survival of the ultraviolet-irradiated conidia of Streptomyces griseus. It has since been recognized for a variety of other ultraviolet effects in a large number of species and tissues (3), including interruption of DNA synthesis (4), production of mutations (5), induction of vegetative phage in lysogenic bacteria (6), inhibition of adaptive enzyme formation in yeast (7), spheration of nudeoli in the grasshopper neuroblast (8), delaying of cleavage in eggs (9), and delaying of division in protozoa (10). Its detailed mechanism is unknown. A possible outline of the process, however, is suggested by existing evidence. * Present address:

A chemically defined medium (MI0) is described in which cells of Haemophilus influenzae Rd grow rapidly (generation time, 35 4 5 min) and reach a stationary level of 1010 cells/ml. Our strain of cells grown in this medium developed high levels of competence when transferred to another medium designed for that purpose. Conditions governing the total development of competence in this organism have now been defined.

A chemically defined nongrowth medium was developed for the induction of competence of Haemophilus influenzae by a stepdown procedure. Cells grown logarithmically in Heart Infusion Broth became competent after being transferred to a medium which consisted of amino acids, sodium fumarate, and inorganic salts. Chloramphenicol (2 ,Ag/ml) or L-valine (1 ,ug/ml) in the nongrowth medium inhibited development of competence. The inhibitory action of L-valine was reversed by comparable concentrations of L-isoleucine. Kinetic studies of the development of competence showed a variable capacity of competent cells to take up deoxyribonucleic acid and reaffirmed earlier findings that competence was not transmissible in H. influenzae. Addition of nicotinamide adenine dinucleotide, thiamine, calcium pantothenate, uracil, and hypoxanthine to the medium for competence resulted in a minimal growth medium in which reduced levels of competence were developed.