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 phage OH or from' the plasmid pHHl together with a DNA fragment conferring resistance to mevinolin. H. halobium P03, a strain lacking pHHI as well as the restriction endonuclease activity found in wild-type H.halobium, was used as the recipient strain. AlR 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 inHf. vokanfi, 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) (8), and the second replicon is derived from the endogenous 150-kbp plasmid pHH1 of H. halobium. A common region of4.3-kbp DNA was determined for all pHH1-type plasmids during the analysis of various deletion derivatives (9). One of the two smallest derivatives, pHH9 (5.7 kbp), is found as a major plasmid, whereas the other one, pHH8 (6.3 kbp), is always present at a 1:1 ratio together with the parental plasmid pHH4 (10).In this paper, we describe the transformation of various H. halobium strains as well as Hf. volcanii with plasmid constructs containing the Hf. volcanii mevinolin-resistance gene combined with these putative H. halobium replicons. As the first application, the smallest H. halobium vector, pUBP2, and the Hf. volcanii vector pWL102 were used to study gas vesicle synthesis. MATERIALS AND METHODS Materials

Structural characteristics of halobacterial gas vesicles

“…In the case of the halophilic archaea, transformation experiments confirmed that the 14 different genes clustered in a region (the vac region) of approximately 9 kb are sufficient for the formation of gas vesicles (Vac+ phenotype) in the Vac-negative recipient, Haloferax volcanii (Blaseio & Pfeifer, 1990;Englert et al, 1992a, b). Hf.…”

“…Shuttle vectors are available that harbour genes conferring resistance to mevinolin (Lam & Doolittle, 1989;Blaseio & Pfeifer, 1990) or novobiocin (Holmes et al, 1991), and compatible origins of replication. The vectors used for complementation studies with the Vac-recipient Hf.…”

Archaeal genetics — the third way

“…Hfx. volcanii also has a restriction system that recognises adenine-methylated GATC sites (which occur frequently in vectors based on E. coli plasmids), resulting in DNA fragmentation followed by plasmid loss or chromosomal integration by recombination 63 . This can be circumvented by passaging the DNA through an E. coli dam -strain that is deficient in GATC methylation 64 .…”

Transcription of the halophage ΦH repressor gene is abolished by transcription from an inversely oriented lytic promoter

“…1, according to Table 1. The PRC products were ligated to the halobacterial shuttle vector pUBP2, which has been described previously [12]. The vector was a gift from F. Pfeifer, Martinsried.…”