Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100

Abstract

To study the functions of the 13 gyp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gyp gene cluster and introduced a kanamycin resistance (K) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gyp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the c cassette in any of the gyp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the K cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the K insertions resulted in phenotypic reversion. Electron microscopic analysis of the K mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicles than in the wild type, while interruptions ofgvpF, -G, -H, -I, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate that gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL,

Gas vesicles in actinomycetes: old buoys in novel habitats?

“…These gas vesicle proteins (Gvps) are encoded by a cluster of gvp genes ( Figure 1). Early gene disruption experiments indicated that 13 gvp genes are essential for wild-type gas vesicle formation in halophilic archaea [3], whereas the findings of a more recent study suggest that eight genes, gvpFGJKLM-gvpAO, suffice [4]. GvpA represents the major envelope component.…”

Formation of a Distinctive Complex between the Inducible Bacterial Lysine Decarboxylase and a Novel AAA+ ATPase

“…Also, the CbbQ gene product, a MoxR-related protein, was shown to alter Rubisco 4 conformation and stimulate its activity upon overexpression of recombinant forms of the proteins together in an Escherichia coli system (13,14). Yet another MoxR family member, GvpN, has been shown to be important in gas vesicle biogenesis in certain organisms (15)(16)(17)(18). The exact role and mechanism of function of MoxR proteins in all of these processes are not clear.…”

GvpD-induced breakdown of the transcriptional activator GvpE of halophilic archaea requires a functional p-loop and an arginine-rich region of GvpD

“…Fourteen gvp genes are involved in the formation of these gas-filled proteinaceous particles, arranged as two oppositely oriented clusters, gvpACNO and gvpDEFGHIJKLM (Englert et al, 1992a;DasSarma et al, 1994). Hbt.…”