A previously undescribed pathway for pyrimidine catabolism

Abstract

The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [(14)C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA-G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively.

Fig. 1.

Pyrimidine catabolic pathways. Known reductive (1) and oxidative (2) pathways for catabolism of pyrimidine rings (A, upper and lower, respectively) and the pathway described in this work (B). Note that ureidomalonic acid and ureidomalonase are not analogous to ureidopropionic acid and ureidopropionase and should probably be renamed.

Fig. 2.

Respiration by wild-type (A) and a strain carrying a lesion in the b1012 operon (B) in medium containing various N sources. Biolog Phenotype MicroArray (PM3) plates were inoculated at an OD₄₀₀ of ≈0.012 and incubated for 4 days at room temperature. Apart from differences in respiration with uracil or uridine as N source, a difference with cytosine (F5) was most notable.

Fig. 3.

Growth (A) and cell yield (B) of NCM3722 (wild-type, black circles) and NCM3996 (b1012::Tn5, gray circles) on NH₄Cl (open circles) or uridine (filled circles). (A) Cells were grown with NH₄Cl or uridine (1 mM) as N source and glycerol as C source at 22°C. Doubling times for NCM3722 on NH₄⁺ and uridine were 3.1 and 7.6 h, respectively. The doubling time of NCM3996 on NH₄⁺ was 3.2 h. (B) Final OD₆₀₀ was determined as a function of the initial concentration of N source.

Fig. 4.

Fate of [U-¹⁴C]uridine. Wild-type strain NCM3722 grown on glycerol and NH₄Cl (5 mM) was diluted 1/100 into medium containing glycerol and uridine (0.5 mM) containing radioactive tracer (see Methods). Radioactivity in cell suspensions (dotted circles), in cells (triangles), and in cell supernatants treated with acid to remove CO₂ (crossed squares) was measured as a function of OD₆₀₀, which was measured for a parallel culture that did not contain radioactive tracer. Residual radioactivity in the acid supernatant after both N atoms of the uridine ring had been consumed indicated that a ¹⁴C-labeled waste product was excreted (see text).

Fig. 5.

Identification of the waste product from uridine. (A) Electron impact spectrum of a peak eluted on GC/MS at ≈7.75 min. This peak was found in supernatants of uridine but not NH₄⁺-grown cells. (B) Electron impact spectrum of 3-hydroxypropionic acid from the National Institute of Standards and Technology Library.

Fig. 6.

Northern blot analysis of expression of the b1012 operon (A) and ethidium bromide-stained loading control (B). RNA extracted from wild-type strain NCM3722 and a congenic strain with a mini-Tn5 insertion in b1013, NCM4062, was hybridized with a b1012 probe as described in Methods. Cells were grown on NH₄Cl (NH₄⁺), arginine (Arg), or uridine (Ura) as sole N source. Only the full-length mRNA of ≈5.7 kb is shown. Cells grown on arginine and uridine showed a reproducible pattern of bands with sizes from 5.7 kb to <2.4 kb. The patterns of bands were very similar for these two N sources and the two strains. Relative amounts of full-length b1012 operon mRNA (normalized to NCM3722 on uridine as 100%) were as follows: NCM3722 NH₄⁺, 1%; arginine, 18%; uridine, 100%; and NCM4062 NH₄⁺, 5%; arginine, 96%, and uridine, 85%). These values were not normalized for slight differences in loading (B).