Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes.

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF-M. The 5'-terminus of the p-gvpF-M mRNA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac- species Haloferax volcanii as recipient. Construct delta A containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac- transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac++). Similarly, double transformants containing p-gvpD-M plus p-gvpACNO, or p-gvpG-M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED-ACNO were Vac++. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac-, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF-M plus p-gvpACNO (delta DE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF-M mRNA could be suggested for p-gvpD and the 5'-region of its mRNA.