Isolation and characterization of a mutant of Neisseria gonorrhoeae that is defective in the uptake of iron from transferrin and haemoglobin and is avirulent in mouse subcutaneous chambers.

Iron-uptake mutants of Neisseria gonorrhoeae strain 340 were obtained following treatment with streptonigrin, and one such mutant (Fud14) was characterized. N. gonorrhoeae strain Fud14 was unable to grow with human transferrin or haemoglobin as the sole source of iron, but grew normally with heat-inactivated normal human serum or haemin. Internalization of 55Fe from transferrin by strain Fud14 was only 25% of the parent level. Strain Fud14 (less than or equal to 1 x 10(8) c.f.u.) did not grow in subcutaneous chambers implanted in mice, whereas the parent strain was infective at an ID50 of 4.3 x 10(1) c.f.u. Supplementation of chambers with either normal human serum or haemin resulted in the establishment of strain Fud14 in vivo for at least 240 h post-inoculation. Electroporation of Fud14 with wild-type DNA and selection for growth on medium containing human transferrin resulted in a recombinant (Fud15) that was capable of utilizing haemoglobin, and was virulent in mice. These results suggest that a gonococcal strain defective in the ability to utilize in vivo iron sources is not capable of survival in vivo.

Environmental factors affecting toxic shock syndrome toxin-1 (TSST-1) synthesis.

The production of toxic shock syndrome toxin-1 (TSST-1) was studied in batch and continuous culture of Staphylococcus aureus strain 1169 in a carbohydrate-free chemically defined medium (CDM). In continuous culture oxygen- and arginine-limitation were required for steady-state TSST-1 synthesis. Aeration suppressed toxin synthesis. The amount of TSST-1 per mg dry weight (specific toxin) at dilution rates from 0.05 to 0.15 h-1 was inversely proportional to the dilution rate. Protease activity increased with increasing dilution rates. In batch culture, TSST-1 began to accumulate in the medium towards the end of the exponential phase of growth, after a critical cell mass was attained. Maximum specific toxin production was observed in medium with an initial pH between 6.5 and 7.0. Growth and toxin synthesis took place in anaerobic conditions when CDM was supplemented with pyruvate and uracil. The Mg++ concentration had no effect on the specific toxin in anaerobic conditions. In aerobic conditions, specific toxin increased c. 23-fold as the Mg++ concentrations increased to 0.4 mM. Further increases in the Mg++ concentration resulted in a reduction in specific toxin.

Identification of an iron-regulated 37,000-dalton protein in the cell envelope of Neisseria gonorrhoeae.

We examined the outer membrane proteins which appear during the growth of Neisseria gonorrhoeae F62 in complex medium supplemented with 25 microM Desferal mesylate, a potent iron chelator. Outer membranes were prepared by Sarkosyl extraction and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several higher-molecular-weight (74,000 to greater than 94,000) proteins increased under iron-limiting conditions. In addition we observed the appearance of an iron-regulated protein with an apparent molecular weight of 37,000. This protein comigrated with the gonococcal protein I under normal Laemmli gel conditions. By increasing the ionic strength of the lower gel buffer, separation of protein I and the 37,000-dalton iron-regulated protein occurred. The 37,000-dalton protein stained poorly with Coomassie blue. However, when a silver stain was used, the protein appeared as a major component of the gonococcal outer membrane. Production of this 37,000-dalton protein was suppressed by the addition of iron to the medium. An iron-regulated protein with a similar molecular weight was observed in four clinical isolates and in an additional laboratory strain. Peptide mapping indicated that the 37,000-dalton protein was distinct from protein I and was identical between strains of the WI and WII serogroups.