Growth and conversion to the mucoid phenotype by nonmucoid Pseudomonas aeruginosa PAO1 was studied in a chemostat system under conditions designed to reflect those likely to be present during chronic infection in the lung in cystic fibrosis patients. Mucoid variants were consistently isolated during continuous culture in the presence of 0.3 M NaCl or 5 or 10% glycerol. Mucoid subpopulations were also detected under conditions of carbon, nitrogen, or phosphate limitation. During carbon or nitrogen limitation, mucoid conversion was dependent upon the choice of substrate. Phosphate-limited cultures exhibited an inverse relationship between culture growth rate and number of mucoid organisms detected. Mucoid variants were not detected when dilution rates (D) exceeded 0.173 h-'. Conversely, at a D of 0.044 h-1, 40% of the population expressed the mucoid phenotype. Phosphorylcholine, a product of phosphojipase C activity on the major lung surfactant phosphatidylcholine, was also used as a growth substrate in nutrient limitation studies. Under all conditions, growth of PAO1 supplied with phosphorylcholine resulted in isolation of mucoid variants, indicating that the lung may provide at least one nutrient source conducive to mucoid conversion. Continuous culture also resulted in detection of a phage associated with strain PAO1. High titers of phage were present under all conditions, including those which yielded no mucoid organisms, suggesting that environmental conditions rather than the phage regulated the appearance of mucoid variants.Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with chronic pulmonary infections in cystic fibrosis (CF) patients (10,15,24). With prolonged chronic infection, P. aeruginosa undergoes alteration from the classical nonmucoid form to an atypical mucoid form, which correlates with the production of the exopolysaccharide alginic acid (6). In the context of chronic lung infection, alginate is an important virulence determinant. The appearance of alginate-producing P. aeruginosa strains is usually associated with a poor patient prognosis (10). Alginate has been implicated in adherence to tracheal epithelium (21, 28) and has been associated with antiphagocytic function (29). While the relationship between mucoid P. aeruginosa and CF is well documented, the environmental factors of the CF lung responsible for conversion of nonmucoid P. aeruginosa to the alginate-producing mucoid form have yet to be defined.A chemostat model for the study of this organism under chronic diseaselike growth conditions (phosphate and nitrogen limitation) which would reflect the likely physiological status of organisms in the lungs of CF patients has recently been reported (18). In this study, phosphate-limited cultures of mucoid P. aeruginosa were capable of utilizing phosphorylcholine (PC), a product of phospholipase C activity on phosphatidylcholine (a major component of lung surfactant), as a sole source of phosphate for growth (18).In an effort to understand the environmen...

Phosphatidylcholine, the major component of lung surfactant, when supplied as the sole source of phosphate for Pseudomonas aeruginosa PAO1, resulted in conversion of as much as 2% of the population to the mucoid phenotype under continuous culture conditions over a 24-day culture period. In addition, growth in phosphatidylcholine resulted in the highest yields of extracellular alginate compared with other environmental conditions. Iron limitation, another environmental condition relevant to the lungs of patients with cystic fibrosis, also resulted in conversion to mucoid. Since both conditions suggested the likelihood of an energy-deprived growth environment as a common variable, the effect of direct inhibition of energy generation by N,N'-dicyclohexylcarbodiimide or gramicidin on the conversion of nonmucoid P. aeruginosa to the mucoid phenotype was examined. Both inhibitors resulted in mucoid subpopulations (0.5 and 0.8%, respectively). Severe energy stress imposed by the combination of phosphate limitation and N,N'-dicyclohexylcarbodiimide treatment resulted in conversion of 55% of the population to mucoidy during a 7-day growth period. A growth advantage of the mucoid over the nonmucoid phenotype was observed under severe nutrient deprivation by growth on unsupplemented Noble agar or in a 1/2,500 dilution of a chemically defined medium. These results clearly demonstrate a significant role for the energy state of the cell in conversion to mucoid and in selection for the mucoid phenotype.

Intracellular and extracellular cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels were measured during the growth of a streptomycin-producing strain of Streptomyces griseus. Increasing the inorganic phosphate concentration of the defined media resulted in a decreased streptomycin titre and alterations in the magnitude and timing of changes in cyclic AMP concentration. Addition of excess inorganic phosphate after 24 h of growth reduced steptomycin production but had no effect on the intracellular cyclic AMP concentration. The results do not suggest that the effect of inorganic phosphate on streptomycin biosynthesis is mediated by cyclic AMP.

Optimum conditions of pH, temperature and multiplicity of bacteriophage infection for the production of phage-associated lysin from Group C streptococci were defined. Lysin obtained by the optimum method was used to make protoplast membranes of Group A type 12 streptococci. These membranes consistently contained very low concentrations of rhamnose, indicating minimal cell wall contamination. The development of such a reproducible technique for the preparation of protoplast membranes will permit more meaningful studies on the involvement of these membranes in the pathogenesis of acute post-streptococcal glomerulonephritis.