The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

ABSTRACT The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3′ portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3′ end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3′ end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.

Investigation of differences between wheat and barley forms of Wheat dwarf virus and their distribution in host plants

Wheat dwarf virus, a monogemini virus, infects several cereal species. Until now complete sequence data have been published only for wheat isolates. We cloned the complete DNA of 21 isolates from wheat, barley and Lolium spec. and compared the sequences with published data. Two types of the virus were found as previously described. Degree of entire nucleic acid homology between both isolates was in the range of 84%. The Large Intergenic Region showed most pronounced differences while the RepA gene was most conserved. No intermediate forms were found, though both isolates co-existed in the same hosts. Sequence data lead to the suggestion that they should be referred to as different viruses rather than strains of a virus.

Analysis of Elements Involved in Pseudoknot-Dependent Expression and Regulation of the repA Gene of an IncL/M Plasmid

ABSTRACT Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation ofrepB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region ofrepA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

The repB gene required for production of extracellular enzymes and fluorescent siderophores in Pseudomonas viridiflava is an analog of the gacA gene of Pseudomonas syringae

Two genes, designated repA and repB, are involved in the regulation of the synthesis of extracellular pectate lyase, protease, and alginate in Pseudomonas viridiflava. The repA gene has been shown to encode a protein highly homologous to several bacterial sensors in the two-component regulator family including the LemA of Pseudomonas syringae. In this study, the repB locus, initially identified in a 6.3-kb EcoRI genomic fragment of P. viridiflava, was further characterized. Results obtained from restriction mapping, deletion subclonings, and mini-Mu-LacZ fusions indicated that the repB gene was contained within a 0.8-kb HindIII–PstI region. Sequence analysis of this repB region revealed the presence of an open reading frame, which was predicted to encode a protein similar or identical to the gacA response regulator found in P. syringae and Pseudomonas fluorescens. The repB gene of P. viridiflava also regulated the production of fluorescent siderophores, in addition to the aforementioned extracellular enzymes and alginate. The repB or gacA homologs were detected in the genomes of nine other strains of P. viridiflava, P. fluorescens, and P. syringae included in the study. The data presented here and earlier indicate that the repA/repB gene regulatory system of P. viridiflava is analogous to the lemA/gacA system of P. syringae and P. fluorescens.Key words: response regulator, signal transduction, soft-rot bacteria, enzyme production.