Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis.

Abstract

We developed vector plasmids for the transformation of Halobacterium halobium, using the replicon region from the halobacterial phase .vphi.H or from the plasmid pHH1 together with a DNA fragment conferring resistance to mevinolin. H. halobium P03, a strain lacking pHH1 as well as the restriction endonuclease activity found in wild-type H. halobium, was used as the recipient strain. All H. halobium fragments tested for autonomous replication as well as the Haloferax volcanii vector pWL102 enabled stable plasmid maintenance in this strain. A frequent loss of all vectors (including pWL102) was observed in Hf. volcanii, where > 90% of the mevinolin-resistant colonies obtained after transformation had lost the vector, presumably because of restriction endonuclease activity and concomitant recombination of the mevinolin resistance marker with the chromosome. The expression of gas vesicle-encoding genes (vac) was analyzed by using a 4.5-kilobase-pair (kbp) fragment containing the plasmid-encoded p-vac gene from H. halobium or an 11-kbp fragment containing the mc-vac chromosomal gene from Haloferax mediterranei for transformation experiments with H. halobium and Hf. volcanii. These experiments indicated that the mc-vac fragment contains all information necessary to synthesize gas vesicles, whereas in the case of the smaller p-vac fragment, complementation by other genes was required for a Vac+ phenotype.