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.     

Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformants

The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc- gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei . Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ΔD transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa , gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation.     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.     

Genome-Wide Analysis of Gene Expression in Stationary Phase and Genetic Characterization of Stationary-Phase-Dependent Halocin Gene Expression in the Haloarchaeon Haloferax mediterranei

The stationary phase of microbial growth is a very complex state regulated by various environmental and physiological factors.An intensive study of stationary phase could promote a comprehensive understanding of the complete life cycle of microorganisms,and may provide important insights into their adaptation to harsh and nutrient-depleted conditions.Although the underlying mechanisms have been well-studied in bacteria and yeasts (Herman,2002;Navarro Llorens et al.,2010),less is known about this growth phase in archaea yet.The haloarchaeon Haloferax mediterranei has served as a good model for studying haloarchaeal physiology and metabolism for several decades because of its accelerated growth,remarkable metabolic ability and genomic stability (Han et al.,2012).During stationary phase,H.mediterranei can produce halocin H4 (Cheung et al.,1997),synthesize gas vesicles (J(a)ger et al.,2002),secrete extracellular polysaccharide (Antón et al.,1988) and accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)(Cai et al.,2012).Due to these specific features,we selected H.mediterranei as a model system to investigate the archaeal gene expression and regulation during the stationary phase.     

Evidence for salt-associated restriction pattern modifications in the archaeobacterium Haloferax mediterranei.

DNA restriction pattern modifications were detected when Haloferax mediterranei was grown in low (10%) salt concentrations. After cells were grown again in optimal (25%) salt concentrations, the original pattern was recovered. These salt-associated DNA modifications were revealed with 5% of the 160 DNA fragments cloned and used as probes in hybridization experiments. Patterns obtained when genomic DNA was digested with different restriction enzymes showed that these modifications are related not to insertions or deletions in genome but to modifications of some specific sequences.     

Genetic transformation of a halophilic archaebacterium with a gas vesicle gene cluster restores its ability to float.

The halophilic archaebacterium, Halobacterium halobium, and many other aquatic bacteria synthesize gas-filled vesicles for flotation. We recently identified a cluster of 13 genes (gvpMLKJIHGFEDACN) on a 200-kb H. halobium plasmid, pNRC100, involved in gas vesicle synthesis. We have cloned and reconstructed the gvp gene cluster on an H. halobium-E. coli shuttle plasmid. Transformation of H. halobium Vac- mutants lacking the entire gas vesicle gene region with the gvp gene cluster results in restoration of their ability to float. These results open the way toward further genetic analysis of gas vesicle gene functions and directed flotation of other microorganisms with potential biotechnological applications.