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

We developed vector plasmids for the transformation of Halobacterium halobium, using the replicon region from the halobacterial phage 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.

Transposition burst of the ISH27 insertion element family in Halobacterium halobium.

Investigation of the plasmid pHH4 in single colonies of Halobacterium halobium PHH4 indicated transposition of insertion elements in 20% of the colonies. Seven ISH27 insertions were observed as well as one ISH23 insertion. The various copies of ISH27 were compared to the two ISH27 elements already present in pHH4, and to the ISH27 element that was identified in the bacteriopsin (bop) gene of a Bop mutant. These ten copies of ISH27 constitute three types on the basis of DNA sequence identity: ISH27-1 (1398 bp), ISH27-2, and ISH27-3 (1389 bp each). The DNA sequence comparison between the three types indicates a region of 1200 bp where the identity between ISH27-1 and ISH27-2 or ISH27-3 is 82-83%. ISH27-2 and ISH27-3 are 95% identical in this region. The remaining region exhibits a lower DNA similarity (64-74% identity) between the different copies. An open reading frame of 1167 nucleotides spans the more conserved region, and a corresponding transcript could be detected in H. halobium PHH4, but not in H. halobium wild-type. ISH27-1 is 91% identical to members of the insertion sequence-like elements ISH51 of Haloferax volcanii, whereas the other two ISH27 element types are 82-83% identical to ISH51. The transposition 'burst' of ISH27 was only seen after storage of the cells for more than two years at 4 degrees C. Upon continuous cultivation at 37 degrees C no transposition event could be observed, suggesting that stress factor(s) might have caused the high transposition rate.

Insertion elements and deletion formation in a halophilic archaebacterium.

Deletion events that occur spontaneously in 36-kilobase-pair (kbp) plasmid pHH4 from the archaebacterium Halobacterium halobium were investigated. Four different deletion derivatives with sizes ranging from 5.7 to 17 kbp were isolated. Three of these deletion variants derived from pHH4 (pHH6 [17 kbp], pHH7 [16 kbp], and pHH8 [6.3 kbp]), whereas the 5.7-kbp plasmid pHH9 derived from pHH6. Strains containing pHH6, pHH7, or pHH9 each lacked the parental plasmid pHH4, while pHH8 occurred at a 1:1 ratio together with pHH4. Common to all of these plasmids was the 5.7-kbp region of pHH9 DNA. The regions containing the fusion site in the deletion derivatives were investigated and compared with the corresponding area of the parental plasmid. Each deletion occurred exactly at the terminus of an insertion element. In pHH6 and pHH7, a halobacterial insertion element (ISH2) was located at the deletion site. The DNA fused to ISH2 displayed a 7-base-pair (bp) (pHH7) or 10-bp (pHH6) sequence homology to the inverted repeat of ISH2. In the two smaller plasmids, pHH8 and pHH9, an ISH27 element was located at the deletion site. Most likely, all of these smaller plasmids resulted from an intramolecular transposition event. The ISH27 insertion sequence contains a 16-bp terminal inverted repeat and duplicates 5 bp of target DNA during the transposition with the specificity 5'ANNNT3'. Four ISH27 copies were analyzed, and two ISH27 element types were identified that have approximately 85% sequence similarity. The ISH27 insertion elements constitute a family which is related to the ISH51 family characterized for H. volcanii, another halophilic archaebacterium.

Dynamic plasmid populations in Halobacterium halobium.

Deletion events occurring in the major 150-kilobase-pair (kb) plasmid pHH1 of the archaebacterium Halobacterium halobium were investigated. We found four deletion derivatives of pHH1 in gas-vacuole-negative mutants, two of which (pHH23) [65 kb] and pHH4 [36 kb]) we analyzed. Both plasmids incurred more than one deletion, leading to the fusion of noncontiguous pHH1 sequences. pHH23 and pHH4 overlapped by only 4 kb of DNA sequence. A DNA fragment derived from this region was used to monitor the production of further deletion variants of pHH4. A total of 25 single colonies were characterized, 23 of which contained various smaller pHH4 derivatives. Of the 25 colonies investigated, 2 had lost pHH4 entirely and contained only large (greater than or equal to 100-kb) minor covalently closed circular DNAs. One colony contained the 17-kb deletion derivative pHH6 without any residual pHH4. The sizes of the pHH4 deletion derivatives, produced during the development of a single colony, ranged from 5 to 20 kb. In five colonies, pHH4 was altered by the integration of an additional insertion element. These insertions, as well as copies of the various insertion elements already present in pHH4, presumably serve as hot spots for recombination events which result in deletions. A second enrichment procedure led to the identification of colonies containing either a 16-kb (pHH7) or a 5-kb (pHH8) deletion derivative of pHH4 as the major plasmid. pHH8, the smallest plasmid found, contained the 4 kb of unique DNA sequence shared by pHH23 and pHH4, as well as some flanking pHH4 sequences. This result indicates that the 4-kb region contains the necessary sequences for plasmid maintenance and replication.

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants.

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.