Genes of de novo pyrimidine biosynthesis from the hyperthermoacidophilic crenarchaeote Sulfolobus acidocaldarius: novel organization in a bipolar operon

Abstract

Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3' end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).

FIG. 1.

De novo synthesis of UMP. Enzymes and their corresponding gene symbols are indicated.

FIG. 2.

Schematic drawing of the pyrimidine gene cluster of S. acidocaldarius and surrounding genes. Grey boxes represent pyrimidine biosynthetic genes. Cross-hatched boxes indicate potential ORFs with unknown function (orf2 to orf8) and the transcriptional regulator Sa-Lrp. Arrows represent mRNA molecules and indicate the start, approximate stop, and orientation of transcription. Positions of the recognition sites for the restriction enzymes EcoRI, SacI, and PstI are indicated (in nucleotides).

FIG. 3.

Nucleotide sequences of the border regions of adjacent genes in the divergent pyrimidine gene cluster. Potential start codons are indicated in bold capitals and are overlined; stop codons are indicated in bold capitals and underlined. Potential SD sequences are indicated in bold capitals.

FIG. 4.

(a) Schematic presentation of the bipolar pyrimidine operon. The approximate position of oligonucleotides used as primers in the RT-PCRs and their polarity are indicated by small arrows. (b, c, d, and e) Analysis by agarose gel electrophoresis of double-stranded DNA fragments generated in the RT-PCRs performed with S. acidocaldarius total RNA, Expand reverse transcriptase (when indicated), Pfu DNA polymerase, and different combinations of primers, as indicated. MW, molecular size markers.

FIG. 5.

(a) Quantitative determination of pyrB transcripts and mapping of the potential transcription initiation site by primer extension. Lanes 1 and 2, pyrB primer extension reactions with 100 μg of total RNA extracted from S. acidocaldarius cells grown in minimal medium supplemented with uracil (50 μg/ml) and minimal medium, respectively, and arrested in the exponential phase. Lanes 7 to 9, primer extension reactions with 100 μg of total RNA from independent cultures grown on complex medium, minimal medium, and minimal medium supplemented with uracil, as indicated. G, A, T, and C, chain terminating DNA sequencing reactions of the noncoding strand obtained with the same ³²P-labeled oligonucleotide used to perform the extension reactions. (b) Mapping of the potential transcription initiation site of pyrE by primer extension. The extension reaction was performed with 100 μg of total RNA extracted from cells grown in minimal medium. (c) Mapping of the potential transcription initiation site located within the pyrC gene. The extension reaction was performed with 100 μg of total RNA extracted from cells grown in minimal medium. (d) Nucleotide sequences of the pyrE-pyrB intergenic region and of the potential internal promoter located in the pyrC coding region. Arrows indicate the start site and orientation of transcription. Potential TATA promoter elements are boxed. (d) Comparison of the pyrE-pyrB intergenic regions of S. acidocaldarius, S. solfataricus, and S. tokodaii. Sequences have been aligned with respect to the strictly conserved TATA box elements. The most important direct repeats (black bars) and imperfect palindromic sequences (grey bars) present in the S. acidocaldarius sequence are indicated. Translation initiation codons are in bold capitals (complementary strand for pyrE), and arrows indicate the start sites and orientations of transcription.