High-frequency mutations in a plasmid-encoded gas vesicle gene in Halobacterium halobium

Gas vesicle-deficient mutants ofHalobacterium halobium arise spontaneously at high frequency (about 1%). The mutants are readily detected, forming translucent colonies on agar plates in contrast to opaque wild-type colonies. To investigate the mechanism of this mutation, we recently cloned a plasmid-encoded gas vesicle protein gene,gvpA , fromH. halobium . In the wild-type NRC-1 strain thegvpA gene is encoded by a multicopy plasmid of ≈150 kilobase pairs (kb). We have now characterized 18 gas vesicle-deficient mutants and 4 revertants by phenotypic and Southern hybridization analyses. Our results indicate that the mutants fall into three major classes. Class I mutants are partially gas vesicle-deficient (Vacδ- ) and unstable, giving rise to completely gas vesicle-deficient (Vac- ) derivatives and Vac+ revertants at frequencies of 1-5%. The restriction map of thegvpA gene region in class I mutants is unchanged but the gene copy number is reduced compared to the Vac+ strains. Class II mutants can be either Vacδ- or completely Vac- but are relatively stable. They contain insertion sequences within or upstream of thegvpA gene. A Vac- class II mutant, R1, contains the 1.3-kb insertion sequence, ISH3, within thegvpA gene, whereas four Vacδ- class II mutants contain other insertion sequences upstream of the gene. Class III mutants are stable Vac- derivatives of either the wild-type or class I mutants and have no detectable copies of thegvpA gene. Based on these results, we discuss the mechanisms of gas vesicle mutations inH. halobium .