Analysis of representative strains of the eight human serotypes of Ureaplasma urealyticum by polyacrylamide gel electrophoresis identified 36 to 40 polypeptides for each strain. At least 80% of the peptides were common among strains, but unique major peptides were identifiable in ureaplasmic types. Type 1 had a polypeptide of 85,000 daltons, type 3 had a polypeptide of 72,000 daltons, type 5 had a polypeptide of 64,000 daltons, and type 8 had a polypeptide of 95,000 daltons. The unique polypeptides in types 1 and 8 were identified as membrane components. Two common major components of 44,000 and 70,000 daltons were observed. Several components were common to some, but not all, serotypes.Patterns obtained from U. urealyticum strains were strikingly different from the patterns of Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, and Mycoplama hominis. Isoelectric focusing demonstrated a unique membrane protein for type 1 at pK 6.4, whereas type 8 possessed an assembly of five unique proteins at pK 7.0. Ureaplasmata were strongly similar to each other by isoelectric focusing, but strikingly different from members of the other genera studied. Although a filtered, strongly buffered dialysate medium with 1% serum and 30 mM urea was used both to maximize yields and to minimize contamination, minor contaminants were detected, which comigrated with horse transfenin (pK 6.0) and cytochrome c (molecular weight, 14,000). The similarities of the polypeptide patterns of U. urealyticum strains affirm their close relationships to each other, in contrast to the diversity shown in the genus Mycoplama, and our recognition of type-specific membrane peptides will enhance the identification of serotypes and the classification of strains.The "T" Mycoplasma strains, isolated from humans, have been classified as a single species, Ureaplasma urealyticum, with eight serotypes (23). The basis for differentiation of the serotypes was the fact that strains of U. urealyticum can be typed reasonably specifically by the metabolic inhibition test or by growth inhibition on agar. However, the metabolic inhibition test shows much greater species specificity than type specificity in the differentiation of Mycoplasma species. Thus, we really do not know whether types of U. urealyticum are as similar to each other as strains of a classic Mycoplasma species or whether the relationship between types is more like that observed between Mycoplasma species which are serologically related (7).The identification of Mycoplasrna species and the determination of the relationships among strains of a species and of related species have been carried out by polyacrylamide gel electrophoresis; patterns from related organisms are quite similar, and those from unrelated organisms are markedly dissimilar (17). The purpose of the present study was to compare the polypeptides and proteins of representative strains of the eight serotypes of U. urealyticum by examining both molecular weights (sodium dodecyl sulfate [SDS ]-polyacrylamide gel electrophor...

The effects of catalase and reducing agents on the growth of Ureaplasma urealyticum type VIII in broth cultures were studied, and used medium was examined for the presence of toxic factors. Growth of U. urealyticum was slower in aged or pre-aerated medium than in fresh medium, although equivalent final yields were achieved. Generation times were 2.9 hr in pre-aerated broth and were 1.34 hr in pre-aerated broth supplemented with either 2mM dithioerythritol or catalase (300 units/ml). Growth and death of U. urealyticum in cultures rendered the broth medium deficient for growth when reinoculated. Filtration of spent medium, adjustment of pH, addition of urea, and reinoculation permitted growth at the same rate and to the same titer as that observed in unused medium. Omission of filtration allowed growth but the final yield was less than that in filtered medium. Addition of sonicated dead cells to filtered, supplemented, spent medium also resulted in only poor growth. Therefore, spent medium appears toxic because it contains organisms (either while or disrupted) that hydrolyze the urea added and again render the medium deficient for growth by depleting an essential growth factor.

The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (j) and the maximum yield ofthe culture, with concentrations of80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4' and was neither a result of increased C1concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCI, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5to 10-fold lower peak yield of organisms. Both A. laidlawii and M. hominis were less sensitive to increased osmotic pressure.

Polyacrylamide gel isoelectric focusing (PAGIF) in thin layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp., and eight strains of Ureaplasma urealyticum (T-strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells. PAGIF was performed in the range of pH 3 to 10. Protein patterns were carefully compared, demonstrating resolved and distinguishable species-specific protein bands. The eight serotypes of U. urealyticum (T-strain) gave identical protein patterns in the pH 3 to 10 range. The characteristic "fingerprints" of a species appeared to correlate with the biochemical nature and not the habitat in each case. Arginine-hydrolyzing species seemed to show more diverse focusing than those that ferment glucose, or prefer an acid environment. Characterization and identification of highly resolved species-specific proteins, ease of performance, and reproducibility of this method suggest that PAGIF might be employed as a taxonomic aid.

The mitogenic property of supernatants from M. arthritidis cultures (MAS) is shown to be nonsedimentable, nondialyzable, labile to 56 degrees C for 1 hr, and active against spleen cells from both normal and germfree mice. Both viable M. arthritidis and MAS were active for T lymphocytes because anti-Thy-1 antiserum and C eliminated responsiveness. Spleen cells enriched for T lymphocytes by passage over nylon columns lost responsiveness unless supplemented with a radioresistant adherent cell population that was shown to bear Ia antigens. Evidence is also presented that the genetic control of T lymphocyte responses to the mycoplasma mitogen was exercised at the level of the Ia-bearing adherent cells. Thus adherent cells from positive responder mouse strains, but not those from nonresponder mouse strains, restored the responses of T cells from F1 hybrids between responder and nonresponder strains.