Development of Competence of Haemophilus influenzae

Abstract

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.

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae

“…Since the chloramphenicol acetyltransferase gene of pACYC184 is known to function in H. in£uenzae [8,11], this plasmid was selected as the source of the CM resistance marker. To determine if the spectinomycin adenyltransferase AAD(9) gene on pSF148 [12] was functional in H. in£uenzae, competent H. in£uenzae strain HI689 [9] was transformed with pSF148 DNA, and transformants were selected on sBHI containing SP. Transformants were selectable on concentrations of SP ranging from 100 Wg ml 3I to 500 Wg ml 3I .…”

“…Each cassette was excised from the vector and inserted by ligation into di¡erent pieces of H. in£uenzae genomic DNA which had been previously cloned in E. coli. The resulting mutagenic construct was con¢rmed by enzyme restriction and subsequently transformed into competent H. in£uenzae [9]. Mutant H. in£uenzae were selected on sBHI under antibiotic selection as described below.…”

Intercellular Effects on Development of Competence in Bacillus subtilis

“…. S<./A/ 5 influenzae also led to the conclusion that no transfer occurs during normal development of compe-ar~~~~~~~~~tence (11,25 for which the concentration of activator substance is proportional to the number of competent cells (33). Consequently, the rate of competence development in Diplococcus increases significantly at high cell densities (32).…”

“…Thus, experiments were carried out to determine whether competence is transferable from competent to noncompetent cultures through cell-to-cell contact. Noncompetent "detector" cultures were obtained by inoculating a genetically distinguishable strain late enough to insure that it did not reach a competent state by the time the "activator" strain was fully competent (11,25). The two cultures were then mixed together in either medium B or medium A, and the relative levels of transformation in mixed and control detector cultures were examined for enhancement in the presence of activator cells.…”

“…In the Diplococcus transformation system, a competenceactivating protein has been described which similarly can induce competence in noncompetent cells, although in this case the factor is more tightly bound to the cells (18,31,32). Although experiments with Haemophilus have failed to demonstrate any intercellular transfer of competence (11,25), evidence has recently been presented that a low molecular weight metabolite present in the supernatant liquids of highly competent cultures can stimulate competence in poorly competent cells (5).…”