Cleavage properties of an archaeal site-specific recombinase, the SSV1 integrase.

SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae. The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome. The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr(314) is involved in the formation of a 3'-phosphotyrosine intermediate. The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature. A discontinuity was observed in the Arrhenius plot above 50 degrees C, suggesting that a conformational transition may occur in the integrase at this temperature. Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction. The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place. Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site. For the cleavage step, the chemical nature of the base in position -1 of cleavage seems to be more important than its pairing to the opposite strand.

An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.