Nucleotide sequence and mutational analysis of an immunity repressor gene from Bacillus subtilis temperate phage ϕ105

We have identified and sequenced a bacteriophage phi 105 gene encoding an immunity repressor, the first to be characterized from a temperate phage infecting a Gram-positive host. Using superinfection immunity as an assay for repressor function, the phi 105 repressor gene was located within a 740-bp PvuII-HindIII subfragment near the left end of the phi 105 EcoRI-F fragment. We show that the repressor is specified by the 5'-proximal coding sequence of a translationally overlapping gene pair, transcribed from right to left on the conventional phi 105 map. Comparison of its amino acid sequence (146 residues) with that of a large number of Gram-negative bacterial and phage repressors revealed a putative DNA-binding region between positions 20 and 39. The coding region is preceded by a strong Shine-Dalgarno sequence 5' AAAGGAG 3'. Deletion analysis of the 5'-flanking DNA allowed to identify transcriptional control elements. Their structure, 5' TTGTAT 3' at -35 and 5' TATAAT 3' at -10, strongly suggests that the phi 105 repressor gene is transcribed by the major vegetative form of B. subtilis RNA polymerase, as would be expected for an early phage gene

High-efficiency transformation of Agrobacterium tumefaciens with plasmid DNA by electroporation

We describe a very simple and highly efficient procedure for direct transformation of Agrobacterium tumefaciens with plasmid DNA. The protocol is based on high-voltage electropermeabilization of whole cells, and reproducible yields of 107 to 5 x 108 transformants per µg of plasmid DNA are obtained. basically electroporation is performed in 15% polyethylene glycol using an electrical field strength of 12.5 kV/cm. The utility of this transformation method is demonstrated by direct establishment in Agrobacterium tumefaciens of a representative genomic library from the plant Arabidopsis thaliana. Our results offer interesting prospects for the transfer of gene banks and for gene complementation in plants without the use of Escherichia coli as an intermediate host

CHARACTERIZATION OF THE BACILLUS-STEAROTHERMOPHILUS MANGANESE SUPEROXIDE-DISMUTASE GENE AND ITS ABILITY TO COMPLEMENT COPPER-ZINC SUPEROXIDE-DISMUTASE DEFICIENCY IN SACCHAROMYCES-CEREVISIAE

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10(5)/μg of DNA to 10(7)/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination

Thermo-inducible gene expression in Bacillus subtilus using transcriptional regulatory elements from temperate phage φ105

A Bacillus subtilis/Escherichia coli shuttle plasmid vector containing a transcriptionally silent chloramphenicol acetyl transferase gene (cat-86) was constructed by ligation of pPL603 (Williams et al., 1981a) and pUC8 (Vieira and Messing, 1982) at their unique EcoRI sites. Using this “promoter probe” vector we have obtained, by direct Cm resistance selection, a collection of cloned Sau3A fragments from the temperate phage φ105 genome exhibiting promoter activity in B. subtilis. 18 promoter plasmids were subsequently transferred to an acceptor cell containing a functional repressor gene of phage φ 105 inserted into the temperature-sensitive replicon pE194. A represser-controlled promoter was identified on the basis of its ability to confer thermo-inducible Cm resistance. The promoter is located on a 650-bp Sau3A. fragment, mapping within the 3.2-kb EcoRI-F fragment, which also contains the φ105 repressor gene. By assaying cloned subfragments of EcoRI-F for expression of immunity against φ105 infection, the repressor gene could be assigned to a 1.1-kb EcoRI-HindIII fragment, which partially overlaps the promoter fragment. Taken together, these results suggest that, like the cI-coded repressor in coliphage λ, the φ 105 represser interacts with an operator sequence mapping very close to its own gene

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromo-succinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Qβ, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1, Qβ and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage