Genetic Recombination in Fused Spheroplasts of Providence alcalifaciens

Abstract

Spheroplasts of Providence alcalifaciens strain P29 auxotrophs were prepared by combined treatment with glycine and lysozyme-EDTA. About 15% of spheroplasts had areas of cytoplasmic membrane exposed where cell wall was absent. The spheroplasts of different auxotrophs were mixed pairwise and fusion was attempted with polyethylene glycol or nascent calcium phosphate. After spheroplasts had regenerated to bacterial forms selection was made for recombinants. Recombinants arose at frequencies of 3.8 x 10-6 to 1.7 x 10-7 per spheroplast initially present, by both methods of fusion. The frequency was strongly dependent on the number of chromosomal loci used in selection. The possible order of five loci was determined and this corresponded to that on the closely related Proteus mirabilis chromosome. Control experiments excluded possibilities of auxotrophic reversion, conjugation, transformation, transfection or transduction as explanations of the results. Analysis of prototrophic clones yielded stable prototrophs or mixtures of stable prototrophs and stable recombinants. Parental types were not encountered. Unselected markers segregated among recombinants. It was concluded that the formation of recombinant bacteria was due to spheroplast fusion and that only stable products of the very temporary heteroploid state were haploid recombinants. The low frequency of recombination was ascribed to the limited number of spheroplasts with areas of exposed cytoplasmic membrane. , 1978). There were three reasons for our choice. Firstly, in preliminary attempts to prepare spheroplasts by the combined method used (see Methods) the conversion frequency of Providence alcalifaciens strain P29 to spheroplasts was higher I N T R O D U C T I O NFodor

Fusion of Spheroplasts and Genetic Recombination ofZymomonas mobilis

“…To examine whether the prototrophic colonies are true fusants or not, the following experiment was conducted as a control. Approximately l0 9 vegetative cells/ml of the two strains were mixed, spread onto Raffinose-R medium, and cultured for 10 to 14 days; very small colonies appeared. This phenomenon seemed to be a result of the growth of the Z-6-C suc -strain in the presence of the Z-6-C frustrain; as shown in Fig.…”

“…Besides, cell fusion could be regarded as a useful method for strain improvement. There have been many reports indicating protoplast fusion in Gram-positive bacteria such as Bacillus,S) Staphylococcus,6) Brevibacterium 7 ) and Streptomyces,S) but only a few on Gram-negative bacteria; for instance, Coetzee et al 9 ) have reported intraspecific fusion in Providence alcalifaciens, and Rastogi et aUO) in Mycobacterium aurum. Therefore, we decided to attempt spheroplast fusion in Z. mobilis, and obtained fusants at high frequencies.…”

Fusion of spheroplasts and genetic recombination of Zymomonas mobilis.

“…There have been manyreports indicating protoplast fusion in Gram-positive bacteria such as Bacillus^Staphylococcus, 6) Brevibacteriurn1) and Streptomyces^but only a few on Gram-negative bacteria; for instance, Coetzee et al 9) have reported intraspecific fusion in Providence alcalifaciens, and Rastogi et al10) in Mycobacterium aurum. Therefore, we decided to attempt spheroplast fusion in Z. mobilis, and obtained fusants at high frequencies.…”

Computer-assisted chromosome mapping in Staphylococcus aureus by protoplast fusion

“…Preparation of protoplasts 8 Polyethylene glycol-induced protoplast fusion Genetic recombination by protoplast fusion has been described in several procaryotic species including Bacillus subtilis (Schaeffer et al, 1976), Bacillus megaterium (Fodor and Alfoldi, 1976) , Brevibacterium flavum (Keneko and Sakaguchi, 1979), Escherichia coli (Tsenin et al, 1979), Providence alcalifaciens (Coetzee et al, 1979), Staphylococcus aureus (Hirachi et al, 1979(Hirachi et al, , 1980Gotz et al, 1981), several Streptomyces spp. (Hopwood et al, 1977;Baltz, 1978;Godfrey et al, 1978), and lactic streptococci (Gasson, 1980).…”

“…Dissolution of the peptidoglycan layers must be accompanied by removal of the outer membrane. In E. coli (Tsenin et al, 1979) and P. alcalifaciens (Coetzee et al, 1979) protoplasts were made by adding glycine to the culture medium and treating the washed cells with a combination of EDTA and lysozyme.…”