Abstract
Gas vesicle synthesis in Haloferax mediterranei involves several gene products encoded by a 9.4-kilobase pair DNA region (mc-vac region) that contains 13 genes in addition to gvpA encoding the major structural gas vesicle protein. The expression of part of this region, encompassing the genes gvpA, gvpC, gvpN, and gvpO was investigated. These genes are transcribed from a common promoter located upstream of gvpA. Transcripts of 0.34 (gvpA only), 1.8 (gvpA/C), 2.4 (gvpA/C/N) and 3 kilobases (gvpA/C/N/O) were observed, with the gvpA transcript being the predominant mRNA species. The majority of the mRNA formed terminates 64 base pairs downstream of gvpA at the cytosine of the sequence 5' TTTTTC 3'. The synthesis of the GvpA and GvpC proteins was investigated by Western analyses. An antiserum raised against isolated gas vesicles of Hf. mediterranei detects, in addition to gas vesicle fragments, the GvpA protein of the M(r) of approximately 8,000 in lysates derived from different halobacteria or from Escherichia coli expressing gvpA. In samples containing isolated gas vesicles, mainly partially disaggregated gas vesicle fragments hybridize, but a minor amount of monomeric GvpA is also seen. For the detection of the GvpC protein, two versions of the gvpC gene (full length and gvp delta C lacking the 3' part encoding the acidic C terminus) were expressed in E. coli, and the resulting proteins were purified. The two antisera raised against these GvpC versions indicate the expression of gvpC in different halobacteria. By Western analysis, GvpC is also detectable in samples containing isolated gas vesicles demonstrating that GvpC is a second, but minor, gas vesicle structural protein.
Highlights
Gas vesicle synthesis in Haloferax mediterranei in- are gas vesicle free (Rodriguez-Valera et al, 1983; Englert et volves several gene products encoded by a 9.4-kilo- al., 1990)
BoththeN-terminalamino acid approximately 8,000 in lysates derived from different sequence determined for gas vesicle preparations, and acidic halobacteria or from Eschericchoilai expressinggvpA. hydrolysis to analyze the aminoacid composition suggest that
By Western analysis, GvpC is detectable in samples containingisolatedgas vesicles demonstrating that GvpC is a second, but minor,gas vesicle structural protein
Summary
From the Max-Planck-Institut f u r Biochemie, 0-8033 Martinsried, Federal Republic of Germany. The major constituent is the 8-kDa proteinGvpA, a gion, encompassing the genes gvpA, gvpC, gvpN, and relatively hydrophobic protein that cannot be identified as a gvp0 was investigated. These genes are transcribed monomer in gas vesicle preparations by SDS-PAGE’ due to from a common promoter located upstream of gvpA. The labeled probes used for Northern hybridization were a 294-bp HincIIIEcoRIfragment (gupA specific; nucleotides 3737-4031 according to thesequence deposited in the EMBL data library),a 260bp EcoRI fragment (gupC-specific;nucleotides 3320-3581), 233-bp NruI (gupN; nucleotides 2095-2328), or 365-bp DraIIIEagI (gup0; terial gas vesicles which contains four ofrive highly conserved nucleotides 1246-1611) DNA fragments. 0.34 -b indicattehe size of the hybridizing mRNA in kilobases
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