Microbial ureases: significance, regulation, and molecular characterization.

[This corrects the article on p. 403 in vol. 40.].

Degradation of purines and pyrimidines by microorganisms.

Urease activity is a physiological function of many bacteria that enables these organisms to utilize urea as a source of nitrogen. The association of ureolytic bacteria with human or animal hosts varies widely from a commensal relationship as demonstrated with skin microflora, a symbiotic relationship in the gastrointestinal tract, to a pathogenic relationship in the urinary tract. Since similar or identical species of bacteria such as Staphylococcus aureus are found in all three environments, the effect of urease activity on the host must be solely a function of the environment of these organisms. In this review, the importance of urease to bacteria is discussed, identifying the gastrointestinal tract as a major reservoir of ureolytic bacteria and investigating the urinary tract environment and the infectious struvite stone production that often accompanies urease-producing bacteria there. Finally, an infection model is presented which explains the development and growth of these urinary calculi and their remarkable persistence in spite of modern urological treatments.

The Ecology and Pathogenicity of Urease-Producing Bacteria in the Urinary Tract

Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess 15N, that is the excess enrichment of 15N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. 15N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of 15N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of 15N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with 15N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the 15N label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.

Helicobacter pylori utilises urea for amino acid synthesis

The mycoplasmatales and the l-phase of bacteria.

Urea is currently considered to be a requirement for the propagation of T-strain mycoplasmas. We report here the replication of T-strain 960 (ATCC 25023) in media prepared from dialyzed components with added putrescine and allantoin but without added urea, or in dialyzed medium containing small amounts of added urea. The least amount of urea which allowed growth in the medium without allantoin was above 10 μg/ml. The amount of urea estimated to contaminate the added allantoin or putrescine was 5 μg/ml or less, which is insufficient to support T-strain replication. T-strain 960 was grown in the presence of urea and the urease inhibitor acetohydroxamic acid AHA where the organisms multiplied at a slower rate in the presence of AHA than in its absence. Urea hydrolysis occurred with concomitant ammonia accumulation and pH increase in cultures with AHA added.

Growth of T-Strain Mycoplasmas in Medium Without Added Urea: Effect of Trace Amounts of Urea and of a Urease Inhibitor

T-strain mycoplasmas were first reported by Shepard in 1954l and more fully described by him two years later.2 These microorganisms are of interest for a variety of reasons, including the finding that they are associated with 6 6 9 3 % of nongonococcal urethritis (NGU) in males.s.4 These organisms were also found in the urogenital tract of 61% of asymptomatic female sexual contacts of male NGU patients, and the females were therefore thought to be carriers of the d i ~ e a s e . ~ The possibility that T-strains are pathogenic in females as well as in males is suggested by the association of these organisms with reproductive failu r e ~ ~ , ~ and premature In general, considerable circumstantial evidence for T-strain pathogenicity has been accumulated to date, but an etiological role for T-strain mycoplasmas in disease has yet to be firmly established. It is our opinion that such a role may prove difficult to establish or to rule out rigorously until more is known about the fundamental biology of these microorganisms. T-strains remain difficult to culture to high titers despite the important discoveries that they prefer an acid pH9 and appear to require urea for g r o ~ t h . ~ . ’ ~ , ’ ~ The low yields plus the observations that T-strains grown in fluid cultures appear to be surrounded by a tenaceous gelatinous matrix4 and carry nonspecific precipitates with them when centrifuged from growth medial2 make difficult the use of these organisms as antigens in immunological studies. The nature of the material associated with the T-strains has not been determined. The very small size of T-strain colonies on agar was, at first, considered a differentiating property,2 but tiny or “T-form colonies have not been a stable characteristic, since increased COz tension in the environment4 and use of appropriate buffers in solid medial3 have allowed for colonies with some surface growth that approach the size and characteristic “fried egg” morphology of classical mycoplasma colonies. The T-strain preference for an acid pH is uncommon among the known mycoplasma species, but other mycoplasmas (e.g. M. horninis) can also grow in an acid pH range.g The only characteristic now thought to truly differentiate T-strains from other mycoplasma species is a unique capacity to hydrolyze urea. This property is still poorly understood, and we have chosen to investigate it further. In early studies with T-strains, horse serum was found to be a superior native protein enrichment in culture media because of its high urea ~ o n t e n t . ~ Dialysis or urease treatment of the horse serum rendered it incapable of supporting Tstrain growth. If, however, the dialysate or urea was reintroduced into the medium, T-strain growth o c c ~ r r e d . ~ Urea was therefore considered to be necessary for the growth of T-strains and is included in T-strain media formulations as an

The growth of t‐strain mycoplasmas in media without added urea*

Using dialyzing cultures of T-strain mycoplasmas, it was possible to make some observations relevant to the growth and metabolism of these organisms which would not be possible in nondialyzing cultures due to growth inhibition of the organisms by elevated pH and increased ammonium ion concentration in media containing urea. The rate of ammonia accumulation was found to be related to the initial urea concentration in the medium and could not be accounted for by any change in the multiplication rate of the organisms. More ammonia was generated than could be accounted for by the added urea alone, suggesting that an ammonia-producing activity other than urease may be present in T-strain mycoplasmas. Titers above 107 color change units per ml were achieved in dialysis cultures of a T-strain mycoplasma in the presence of urea, and such titers were maintained for approximately 60 h during dialysis culture in the absence of added urea.

Dialysis Culture of T-Strain Mycoplasmas

SUMMARY: The rate of accumulation of ammonium ion in cultures of Ureaplasma urealyti-cum was independent of the growth rate and of the initial urea concentration above 0.025% in the medium, although the quantity of ammonium ion accumulating did depend on the initial urea concentration. Ammonium ions accumulated at a similar rate in U. urealyticum cultures of both rapidly and slowly growing organisms. Viable but non-growing ureaplasmas also produced ammonia in complete medium at a lower temperature than usual (25°) or in an inadequate growth medium at 37 °. The rate of ammonium ion accumulation in a dying culture depended on the number of viable organisms present; this is relevant to diagnostic methods for ureaplasmas which depend on detecting ammonia colorimetrically.

The Effect of Growth and Urea Concentration on Ammonia Production by a Urea-hydrolysing Mycoplasma ( Ureaplasma urealyticum)

Urealytic activity of the cytoplasmic fraction of Ureaplasma urealyticum prepared by digitonin lysis was assayed in a simple buffer system (HEPES plus EDTA) by measuring the release of 14CO2 from [14C]urea. The Km of this preparation agreed with our previous observations of the same activity measured in a more complex reaction mixture. The substrate concentration at which maximum velocity occurred was approximately 20 mM. The activity was sensitive to heavy metals and inhibitors which react with sulphydryl groups such as N-ethylmaleimide and p-chloromercuribenzoate. It was not inhibited by Ca2+ or Mg2+ or by the reaction products, ammonia and carbon dioxide.

Gerald K. Masover

Papers

Growth of T-Strain Mycoplasmas in Medium Without Added Urea: Effect of Trace Amounts of Urea and of a Urease Inhibitor

The growth of t‐strain mycoplasmas in media without added urea*

Dialysis Culture of T-Strain Mycoplasmas

The Effect of Growth and Urea Concentration on Ammonia Production by a Urea-hydrolysing Mycoplasma ( Ureaplasma urealyticum)

Some characteristics of Ureaplasma urealyticum. Urease activity in a simple buffer: effect of metal ions and sulphydryl inhibitors.