Localization of enzymes in Ureaplasma urealyticum (T-strain mycoplasma).

Ureaplasma urealyticum cells were lysed by osmotic shock or by digitonin. The membrane fraction contained four to ten times as much protein as the cytoplasmic fraction. These values are in large excess of those reported for classical mycoplasmas, suggesting that the Ureaplasma membrane fraction was heavily contaminated with proteins derived from the growth medium. The U. urealyticum urease activity was localized in the cytoplasmic fraction, whereas the adenosine triphosphatase activity was localized in the membrane fraction. Significant urease activity could be detected also in nonviable cells. Urea, at concentrations above 0.25 M, was mycoplasmastatic to Acholeplasma laidlawii, Mycoplasma hominis, and U. urealyticum, so that the Ureaplasma urease did not afford preferential protection against urea toxicity. The intracellular localization of the urease would be expected to release ammonia from urea in the cytoplasm. The ammonia will take up protons to become ammonium ions. It can be hypothesized that the intracellular NH4+ plays a role in proton elimination or acid-base balance, which might be coupled to an energy producing ion gradient and/or transport mechanisms.     

Effects of carbon dioxide, urea, and ammonia on growth of Ureaplasma urealyticum (T-strain mycoplasma).

By use of a simple device for continuous CO2 gassing of Ureaplasma urealyticum cultures growing in a liquid medium, we have been able to separate some of the effects of urea, CO2, ammonia, and pH on growth. The CO2 acted as a superior buffer in the pH range 5.7 to 6.8, which is optimal for Ureaplasma growth. It was, therefore, possible to observe the effect of repeated additions of urea to the culture without alkalinization of the growth medium. We found that the repeated additions of urea did not enhance Ureaplasma growth, and the resultant accumulation of ammonium ions (greater than 2,000 microng/ml) did not cause more rapid death under these conditions. By abruptly changing the gaseous environment from CO2 to N2, it was possible to cause a rapid pH change in the culture to a value above 8.0. This resulted in a more rapid death of the organisms.     

Urea-hydrolyzing activity of a T-strain mycoplasma: Ureaplasma urealyticum.

The urea-hydrolyzing activity of a T-strain mycoplasma was studied in experiments using whole cells and cell-free enzyme preparations by measuring the release of 14CO2 from [14C]urea. Under the conditions used, the urea concentration optimum is approximately 5.6 X 10(-3) M urea. The activity is soluble and not membrane bound. It is stable at -70 C for several weeks but is more labile at higher temperatures. The pH optimum is between 5.0 and 6.0. The effect of several inhibitors on the activity was tested and revealed similarities, as well as differences, between T-strain mycoplasma urease activity and the urease activity of other organisms and plants.     

Cytochemical evidence of extramembranous carbohydrates on Ureaplasma urealyticum (T-strain Mycoplasma).

Cells of Ureaplasma urealyticum that were prepared by a ruthenium red technique demonstrated an extramembranous layer of polyanions of about 15 to 30 nm in width. Application of the concanavalin A-iron dextran strain indicated that the outer surface of this layer contained glucosyl-like residues.     

Biosynthesis of saturated and unsaturated fatty acids by a T-strain mycoplasma (Ureaplasma).

A human T mycoplasma (Ureaplasma urealyticum) incorporated radioactivity into its lipids from [1-14C]acetate in the growth medium. Methanolysis of the lipids showed the label to be confined almost entirely to the methyl esters of the fatty acids. About 80% of the label was associated with the methyl esters of the saturated fatty acids, and the rest was found in the unsaturated methyl ester fraction. Gas-liquid chromatography of the saturated methyl esters showed the label to be present in the peaks of palmitate, myristate, and stearate, whereas in the unsaturated methyl ester fraction most of the radioactivity emerged in the peak of palmitoleate. The addition of either oleic or palmitic acid to the growth medium markedly decreased the organisms' incorporation of radioactivity from acetate. It is concluded that the T mycoplasma strain is capable of de novo synthesis of both saturated and unsaturated fatty acids, in this respect differing from all of the Mycoplasma and Acholeplasma strains investigated to date.     

Effect of fluoropyrimidines on the growth of Ureaplasma urealyticum

In the present study, we investigated the effect of fluoropyrimidines on the growth of Ureaplasma urealyticum. Addition of fluoropyrimidines strongly inhibited bacterial growth. Growth inhibition by these analogues could be reversed by addition of either thymidine or deoxyuridine, suggesting inhibition of thymidylate biosynthesis as the mechanism in operation.     

Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth

Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu-TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu-TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu-TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.     

Effect of Fluoropyrimidines on the Growth of Ureaplasma urealyticum

In the present study, we investigated the effect of fluoropyrimidines on the growth of Ureaplasma urealyticum. Addition of fluoropyrimidines strongly inhibited bacterial growth. Growth inhibition by these analogues could be reversed by addition of either thymidine or deoxyuridine, suggesting inhibition of thymidylate biosynthesis as the mechanism in operation.     

Effect of Urea Concentration on Growth of Ureaplasma urealyticum (T-Strain Mycoplasma)

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 × 10⁴ colony-forming units per ml. The maximum yield of organisms, 8.0 × 10⁷ colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 × 10¹⁰ total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K_s value for growth was 2.0 × 10⁻⁴ M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.     

Ureaplasma urealyticum and Ureaplasma parvum in etiology of urogenital mixt-infections

The complex study of 104 vaginal samples from patients with urogenital uroplasmosis was carried out. U. parvum were detected in 67.3% patients, U. urealyticum--in 12.5% and in 20.1% cases--two species were registered at the same time. Isolation of clinical significant concentration of both ureaplasma (> 10(4) CFU/ ml) was detected in about 50% of cases. Expression of inflammation of vaginal mucus depended on the level of concentration of infection agents. U. parvum were associated with bacterial vaginosis, while in urogenital candidosis U. parvum was detected rarer than U. urealyticum. The dominant numbers of clinical ureaplasma were high sensitive to "new" macrolides and chinolons, however the high percent of isolates were resistant to erytromicin and doxiciclin.     

Impact of Mycoplasma hominis and Ureaplasma urealyticum on the concentration of proinflammatory cytokines in vaginal fluid

The main aim of this study was to determine impact of Mycoplasma hominis and Ureaplasma urealyticum on the concentrations of selected proinflammatory cytokines in vaginal fluid in pregnant women. The samples were obtained from 120 pregnant women at 22 to 36 weeks gestation. Vaginal fluid were analyzed for the concentrations of IL-1 alpha, IL-1 beta, IL-6 and IL-8 using standard enzyme-linked immunosorbent assay technique (ELISA), and cervical fluid for prevalence of Mycoplasma hominis and Ureaplasma urealyticum. Genital mycoplasmas were diagnosed in 36 of 120 pregnant women (30%), (in 17 of 36 women (47.2%) both M. hominis and U. urealyticum, in 14 women (38.9%) only U. urealyticum, and in 5 cases (13.8%) only M. hominis were diagnosed). Vaginal levels of IL-8 was statistically higher among women with genital mycoplasmas infection, as compared to group without these bacteria (p=0.033), while there was no correlation between IL-1 alpha, IL-1 beta and IL-6 concentrations and genital mycoplasmas infection. Future studies should concentrate on evaluation the impact of other lower genital tract bacteria on concentration of IL-8 and other proinflammatory cytokines.     

Equation for osmotic pressure of serum protein (fractions).

The colloid or protein osmotic pressure (Pi) is a function of protein molarity (linear) and of Donnan and other effects. Albumin is the major osmotic protein, but also globulins influence Pi. Equations based on concentrations of albumin and nonalbumin (globulin concentration + fibrinogen concentration) protein approximate Pi better than albumin alone. Globulins have a wide range of molecular weights, and a 1956 diagram indicated that Pi of globulin fractions decreased in the order alpha1-, alpha2-, beta-, and gamma-globulin. The molecular weight of the serum protein fractions had been extrapolated, so van't Hoff's law and nonlinear regression analysis of the curves permitted expression of the diagram as an equation: product Pi(s,Ott,2 degrees C,cmH2O)=x(alb)(0.338C(tot)+0.00339C(tot)(2))+x(alpha1)(0.518C(tot)+0.0107C(tot)(2))+x(alpha2)(0.203C(tot)+0.00155C(tot)(2))+x(beta)(0.187C(tot)+0.000577C(tot)(2))+x(gamma)(0.161C(tot)+0.000223C(tot)(2)), where Pi(s,Ott,2 degrees C,cmH2O) is Pi of serum at 2 degrees C (in cmH2O) computed from the 1956 diagram, C(tot) is the concentration (g/l) of total protein in serum, and x(alb), x(alpha1), x(alpha2), x(beta), and x(gamma) are the fractions of albumin, alpha1-, alpha2-, beta-, and gamma-globulin, respectively. At one and the same concentration of fractions, Pi("Ott") decreases in the order alpha1-globulin, albumin, alpha2-globulin, beta-globulin, and gamma-globulin.     

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains

ABSTRACT: BACKGROUND: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. RESULTS: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.750.78 Mbp genomes and UUR serovars were 0.840.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. CONCLUSIONS: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The 4 overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that 6 ureaplasmas exist as quasispecies rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.     

解脲脲原体耐药性研究进展

解脲脲原体是条件致病菌。目前对其耐药性的研究主要包括对喹诺酮类、大环内酯类和四环素类3种抗菌药物相关耐药突变位点的检测,以及生物膜对病原体相关药物敏感性的影响,研究方法和检验手段仍较为传统、局限,研究方案也仅停留在对前人实验的重复。近年来,有学者将多位点序列分型技术用于解脲脲原体耐药序列类型的研究。在完善耐药机制研究的基础上,如何实现对耐药株的快速鉴定,从而指导抗菌药物的合理选择等仍需进一步研究。     

Morphology of Ureaplasma urealyticum (T-mycoplasma) organisms and colonies.

The morphology of Ureaplasm urealyticum in broth cultures was studied by phase-contrast microscopy. Most organisms appeared singly or in pairs. Long filaments and long chains of cocci, common in classical mycoplasma cultures, were not observed. On solid medium, U. urealyticum produced "fried-egg" colonies which developed according to the scheme suggested by Razin and Oliver (J. Gen. Microbiol., 1961) for the morphogenesis of the classical mycoplasma colonies. The formation of the peripheral zone of the colonies followed that of the central zone only when growth conditions were adequate, Hence, the appearance of peripheral zones, and consequently the larger colony size, can be taken as an indicator of improved growth conditions. Incubation in an atmosphere of 100% CO2 resulted in significantly larger colonies than in an atmosphere of N2, O2, or air. CO2 acts as a buffer, keeping the pH at the optimal range for Ureaplasma growth (pH 6.0 to 6.5) in the presence of the ammonia produced from the urea hydrolyzed by the organisms. The addition to the medium of 0.01 M urea together with 0.01 M putrescine enabled better growth than with urea alone. Small amounts of phosphate improved growth in an atmosphere of CO2, apparently fulfilling a nutritional role. Under nitrogen, higher phosphate concentrations were required for good growth, apparently serving as a buffer as well as a nutrient. Sodium chloride and sucrose which had been added to increase the tonicity of the medium inhibited growth above 0.1 M. An increase in the agar concentration above 2% resulted in decreased colony size. Likewise, prolonged drying of the agar plates caused a marked decrease in colony size, mostly affecting the peripheral zone. The addition of both urea and putrescine to the growth medium and incubation in a humidified CO2 atmosphere are recommended for improved growth and formation of fried-egg colonies of U. ureaplyticum on agar. It must be emphasized that these experiments were carried out with a laboratory-adapted strain.     

Effect of osmotic pressure on root growth,cell cycle and cell elongation

Summary The paper reports a study of root growth, the duration of the cell division cycle and cell size, inAllium cepa roots grown in mannitol solution, with osmotic pressures of 0–16 atm., at 25° C, with aeration. Root growth decreases markedly as the osmotic pressure rises, at 12 atm. being about 35% of what it is at 0 atm. The rate of the cell cycle, , expressed as the percentage of cells passing through any given point in the cycle in one hour, is a roughly linear function of the osmotic pressure, and at 12 atm. is reduced to 80% of what it is at 0 atm. The reaction of the cell size to osmotic pressure is similar to that of the growth. The relative values of the two diverge progressively as the osmotic pressure increases and at 12 atm. the elongation of the cells has dropped to 40% of normal.The data obtained agree with those given by the equationG = K · · L, in whichK is a constant, is the rate of the cycle (reciprocal of its duration) andL the average size of the mature epidermal cells.     

Detection and automatic control of ammonium ion concentration in miceobial culture with an ammonium ion selective electrode

An ammonium ion selective electrode (AISE) had a membrane of polyvinyl chloride in which the antibodies nonactin and monacin were embedded. The detection range was 0.1–200 mM. The step response was 90% in 20 seconds. The output of the AISE increased 6% with a 1°C rise temperature. The output of the AISE was constant between pH 4–7. The selectively coefficient of potassium ion was 0.158 and hence its interferring effect must be considered. The selectivity coeficcients of other cations were small enough to be negligible. Throughout a batch culture of Escherichia coli, values calculated by subtrating (selectivity coefficient) × (potassium ion concentration) from the detected output of the AISE agreed with actual concentrations of ammonium ion. An automatic. constant-value, feebdack control system of ammonium ion concentration was attempted by on-off controlled supply of solution containing both ammonium and potassium ions, the proportion of whose concentration was made equal to the proportion of their average volumetric consumption rates by a microorganism in batch culture. By this control system, ammonium ion concentrations in culture supernatants of fed-batch cultures of Escherichia coli and Saccharomyces cerevisiae could be maintained vitrually at constant levels (5±0.8 mM for the cultivation of E. coli and 50±5 M for the cultivation of S. cerevisiae).