Homologous and heterologous expression of RNase III from Lactococcus lactis.

The endoribonuclease III (RNase III), encoded by the rnc gene, is an important enzyme for RNA metabolism. In this report a chromosomal fragment containing the rnc gene from Lactococcus lactis was cloned and its expression was analyzed. Complementation assays performed in Escherichia coli demonstrate that the lactococcal RNase III (Lac-RNase III) is able to process rRNAs and to regulate the levels of polynucleotide phosphorylase (PNPase). These results demonstrate that the lactococcal enzyme is able to substitute the Ec-RNase III not only in the rRNA processing, but also in the processing of mRNAs. The amount of lactococcal rnc transcript in an E. coli Deltarnc strain was 3.3-fold higher than in the wild type strain, suggesting that the E. coli RNase III triggers the degradation of the heterologous rnc mRNA. Lac-RNase III is able to cleave an in vitro synthesized mRNA substrate specific for the Bacillus subtilis homolog. Using this substrate, we standardized an enzymatic assay which allows the specific detection of the endonucleolytic activity of Lac-RNase III in L. lactis and E. coli crude extracts.

Development of an inducible system to control and easily monitor gene expression in Lactococcus lactis.

This report describes the implementation and use of a maltose-inducible system for regulated gene expression in Lactococcus lactis. The system was established using Green Fluorescent Protein as reporter. The transcription of a gene of interest from the inducible promoter of pLS1RGFP plasmid vector can be easily monitored by fluorescence spectroscopy and microscopy. As an example, the lactococcal ribonuclease III was overproduced in an active form.

Biochemical analysis of point mutations in the 5'-3' exonuclease of DNA polymerase I of Streptococcus pneumoniae. Functional and structural implications.

To define the active site of the 5'-3' exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp(10), Glu(88), and Glu(114), which are conserved among all prokaryotic and eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp(10) in the catalytic event, (ii) a role of Glu(114) in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu(88) for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.

Purification and properties of the 5'-3' exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing.

A D10A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site directed mutagenesis of the polA gene. Introduction of the mutation resulted in a drastic decrease of the 5'-3' exonucleolytic activity present in the wild-type enzyme. Moreover, the mutation at the D10 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for the proposed direct involvement of this Asp residue in a metal-assisted 5'-3' exonucleolytic reaction in type I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases. The D10A mutant polypeptide retained the polymerase activity of its parental enzyme, it is able to incorporate correctly nucleotides in a DNA template, and efficiently uses labeled and unlabeled nucleotides analogues in DNA sequencing by the dideoxy-chain-termination method. These characteristics convert this polymerase into a useful tool for manual and automatic sequencing.

Purification and properties of the 5'-3' exonuclease D190-->a mutant of DNA polymerase I from Streptococcus pneumoniae.

A D190-->A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site-directed mutagenesis of the polA gene. Comparison of the S. pneumoniae DNA polymerase I, its polymerase domain, and the [Ala190]exonuclease mutant revealed that the mutant polypeptide retains the polymerase activity of the parental enzyme and displayed the strand-displacement activity of its polymerase domain. However, introduction of the mutation resulted in a 2500-fold reduction of the 5'-3' exonuclease catalytic rate compared with the wild-type enzyme. Moreover, the mutation at the Asp190 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for a direct involvement of this aspartic acid residue in a metal-assisted 5'-3' exonucleolytic reaction in type-I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases.