Functional studies of the gvpACNO operon of Halobacterium salinarium reveal that the GvpC protein shapes gas vesicles

Abstract

Gas vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the plasmid pHH1-encoded vac (p-vac) region consisting of 14 different gvp genes that are arranged in two clusters, p-gvpACNO and, oriented in the direction opposite to that of gvpA, p-gvpDEFGHIJKLM. The p-gvpACNO region was analyzed at the transcriptional and functional levels in H. salinarium and in Haloferax volcanii transformants containing subfragments of the p-vac region. The p-gvpACNO genes were transcribed as several mRNAs: the 270-nucleotide (nt) p-gvpA transcript, encoding the major structural protein, occurred in large amounts, and minor amounts of three different readthrough transcripts (p-gvpACN, and p-gvpACNO mRNA) were found. In addition, the p-gvpO gene gave rise to two separate mRNA species: a 550-nt mRNA starting at the ATG and spanning the entire reading frame and a 420-nt RNA encompassing the second half of the p-gvpO gene. The requirement of p-gvpC, p-gvpN, and p-gvpO gene expression for gas vesicle synthesis was assessed by transformation experiments using the VAC- species Haloferax volcanii as the recipient. A delta C transformant, harboring the p-vac region with a deletion of the p-gvpC gene, produced large amounts of irregularly shaped gas vesicles. A shape-forming function of p-GvpC was demonstrated by complementation of the delta C transformant with the p-gvpC gene, resulting in wild-type-shaped gas vesicles. In the delta N transformant, the level of gas vesicle synthesis was very low, indicating that the p-GvpN protein is not required for gas vesicle assembly but may enhance gas vesicle synthesis. The p-gvpN deletion did not affect accumulation of p-gvpACO mRNA but reduced the separate p-gvpO transcription. The delta O transformant was Vac- and had a strongly decreased level of p-gvpACN mRNAs, demonstrating that the p-GvpO protein is required for gas vesicle synthesis and may affect transcription of this DNA region.