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

Summary 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 mRMA 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 ΔA 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 (ΔDE) 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.