Specific Protein Differences Among Strains of Ureaplasma urealyticum as Determined by Two-Dimensional Gel Electrophoresis and a Sensitive Silver Stain

Abstract

The proteins of nine strains of Ureaplasrna urealyticum, representing serovars I through IX, were analyzed by using two-dimensional gel electrophoresis and a sensitive silver staining procedure. The electrophoretic patterns of the nine strains tested could be divided into two distinct major groups, one group consisting of serovars 11, IV, V, VII, VIII, and IX (group A) and one group consisting of serovars I, 111, and VI (group B).Ureaplasma urealyticum strains are a serologically heterogeneous group of organisms (16) that are frequently found in human genital tracts. The pathogenic potential of these organisms has been widely disputed, and the significance of their isolation is often unclear (4, 17, 18). The possibility that some strains or biotypes of U. urealyticurn are pathogenic and others are not has been suggested (15), but serological differences have not been clearly associated with pathogenicity (10,15). Deoxyribonucleic acid hybridization (5) and one-dimensional (8, 13) and two-dimensional (9) polyacrylamide gel electrophoretic analyses of [3'S]methionine-labeled proteins have revealed differences among strains of U . urealyticum. In two of these studies (5, 9), the nine recognized serovars could be separated into two major groups. In this report we describe an additional method of analyzing the proteins of U. urealyticurn strains by using two-dimensional electrophoresis and a sensitive silver-based stain which combines with proteins in a fashion that produces reproducible and characteristic colors (12). Our findings confirm previous reports of two groups within the species U. urealyticurn.Eight strains of U . urealyticum originally obtained from F. T. Black, Aarhus, Denmark, and designated serovars I through VIII (3) and one strain obtained from J. A. Robertson, Alberta, Canada, and designated serovar IX (11) were used. These organisms were grown in U-9 broth (14) supplemented with 2% PPLO serum fraction (Difco Laboratories, Detroit, Mich.) at 37°C in an atmosphere containing 5% C02. Cultures in the log phase of growth were harvested by centrifugation at 12,000 x g for 1 h and washed three times with phosphate-buffered saline. The cell pellet obtained from 1 liter of a 20-h culture was sufficient for more than six analyses. Uninoculated medium was incubated and alkalinized to the same degree as growing cultures and was then centrifuged and processed in the same manner. Each strain was grown and tested at least three times.Each washed cell pellet was suspended in six times its volume of sample buffer containing 2% sodium dodecyl sulfate, 2% P-mercaptoethanol, and 1% cyclohexylaminoethanesulfonic acid (Calbiochem-Behring Corp., La Jolla, Calif.) (pH 9.5) and heated at 98°C for 10 min. Electrophoresis in the first dimension (isoelectric focusing) and the second dimension (sodium dodecyl sulfate slab gel electrophoresis) was performed by using the method of Anderson et al. (2), as described by Dunbar et al. (6). Cylindrical isoelectric focusing gels containing ampholines with a wide pH range (pH 3.5 to 10...

Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns

“…One involved types 1 to 9, and the other involved types 1 to 9, 11, 12, and 14 (83,131). Although the serovars could be divided into the two genetic clusters (see below), only one serovar-unique protein was found and this was in serovar 9 (131). All other polypeptides were common to all serovars, limited to one of the two genetic clusters, or found in two or more serovars which were not always of the same group.…”

Phylogenetic Analysis of Genes Coding for 16S rRNA in Mammalian Ureaplasmas

“…The following six species have been recognized in the genus Ureaplasma: Ureaplasma urealyticum, isolated from humans; Ureaplasma diversum, isolated from bovines; Ureaplasma gallorale, isolated from birds; Ureaplasma felinum and Ureaplasma cati, isolated from cats; and UreapZasma canigenitalium, isolated from dogs. The accumulated data suggest that the human species U. urealyticum consists of two different clusters, the T960 and parvo biovars, which can be distinguished on the basis of the results of polyacrylamide gel electrophoresis of cell proteins (12,20), DNA-DNA homology data (1), restriction endonuclease cleavage patterns (13), restriction fragment length polymorphism data (4), manganese susceptibility data (14), and DNA modification system data (2). The genome sizes of U. urealyticum strains have been estimated to be 760 to 1,170 kbp by pulsedfield gel electrophoresis (9).…”

Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum

“…The serovar-specific antigens appear to be on or in the surface membrane and they may be either lipid or proteinaceous [20]. Twodimensional gel electrophoresis has shown conflicting results with serovar-specific proteins, biovar-specific proteins and proteins common to all serovars [21].…”