ABSTRACT
Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37 degrees C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37 degrees C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.
Subject(s)
Alcaligenes/genetics , Chromosomes, Bacterial , Alcaligenes/drug effects , Chromosome Mapping , Conjugation, Genetic , Ethyl Methanesulfonate/pharmacology , Genetic Linkage , Methylnitronitrosoguanidine/pharmacology , Mutagenesis , Plasmids , TemperatureABSTRACT
A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.
Subject(s)
Alcaligenes/genetics , Bacterial Proteins/genetics , DNA Transposable Elements/genetics , Amino Acid Sequence , Base Composition , Base Sequence , Cloning, Molecular , DNA, Bacterial , Molecular Sequence Data , Open Reading Frames , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Sucrose/metabolismABSTRACT
The IncP1 plasmid pULB113 (RP4::miniMu) not only mediates the transfer of chromosomal markers in the classical direction (i.e. from the donor to the recipient cell) but also in the opposite direction (i.e. from the recipient bacterium to the donor). This phenomenon of retrotransfer was observed in homologous matings with Pseudomonas fluorescens, Alcaligenes eutrophus and Salmonella typhimurium. Retrotransconjugants could be discriminated from direct transconjugants by appropriate chromosomal and plasmid markers used to distinguish the mating partners not bearing pULB113. Retrotransfer of chromosomal markers occurred at frequencies equal to, or sometimes greater than, those observed for the direct mobilization, thus allowing the recovery of "recipient" recessive markers in the "donor" with linkage values similar to those found in the normal direction. Retrotransfer was also observed in heterospecific matings involving A. eutrophus and pULB113 bearing P. fluorescens: R-primes carrying different selected and unselected markers were recovered in both bacteria. "Retrotransfer" or "shuttle transfer" seems to be a specific trait of IncP1 plasmids.
