COMPLEMENTATION STUDIES WITH THE GAS VESICLE-ENCODING P-VAC REGION OFHALOBACTERIUM-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 gene s that are arranged in two clusters: p-gvpACNO and, oppositely oriente d, 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 a nd p-gvpK gene was expressed in Escherichia coli and antibodies to pro teins 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 transform ation experiments using the Vac(-) species Haloferax volcanii as recip ient. Construct Delta A containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac(-) tra nsformants, but the addition of p-gvpA on a second vector restored gas -vesicle synthesis to wildtype level (Vac(++)). Similarly, double tran sformants containing p-gvpD-M plus p-gvpACNO, or p-gvpG-M (fused to th e promoter of the halobacterial ferredoxin gene for expression) plus p -gvpFED-ACNO were Vac(++). Transformants containing the p-vac region e ither lacking gvpA, gvpF, or gvpGHI were Vac(-), indicating the absolu te requirement of these gvp genes (or at least one in the case of gvpG HI) for gas-vesicle formation. Double transformants containing the con structs p-gvpF-M plus p-gvpACNO (Delta DE) accumulated gas vesicles (V ac(+)) but synthesized fewer than the wild type, showing that the p-gv pDE genes are not necessary for gas-vesicle assembly. A repressor func tion affecting the synthesis of the p-gvpF-M mRNA could be suggested f or p-gvpD and the 5'-region of its mRNA.