The characterization of the nv- gvpACNOFGH gene cluster involved in gas vesicle formation in Natronobacterium vacuolatum

Abstract

The haloalkaliphilic archaeon Natronobacterium vacuolatum forms cylinder-shaped gas vesicles throughout the growth cycle when grown in media containing 15-25% NaCl. Cells cultivated in media containing 13% NaCl are, however, gas-vesicle-free. The major gas vesicle structural protein, nv-GvpA, was detected by an antiserum raised against the gas vesicles of Haloferax mediterranei; the antiserum reacted with an 8.3-kDa protein in samples containing cell extracts or purified gas vesicles of N. vacuolatum. The gene encoding nv-GvpA was isolated together with six additional gvp genes; these genes are arranged consecutively in a cluster as nv-gvpACNOFGH and are cotranscribed. Transcript analysis by primer extension revealed only one start site three nucleotides upstream of the nv-gvpA reading frame. This arrangement of gvp genes differs from that of the gas-vesicle-encoding genes in Halobacterium salinarium and Hf. mediterranei. The comparison of the deduced Gvp protein sequences indicated similarities with the respective halobacterial Gvp proteins, with GvpA exhibiting the highest degree of conservation (97-100%). The second gas vesicle structural protein, nv-GvpC, was 150-250 amino acids longer than all other halobacterial GvpC proteins and was much less conserved (48-73%). The expression of the nv-gvp genes was monitored in N. vacuolatum cells cultivated in 20 or 13% salt media. Northern and Western analyses showed that despite the lack of gas vesicles in cells grown in 13% salt medium, the gvpACNOFGH gene cluster was transcribed and GvpA protein was synthesized, suggesting that the absence of gas vesicles is not due to a lack of transcription.

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“…This was further confirmed by the observation of laddering of GvpL protein on gels. These genetic and biochemical results are consistent with the findings of comparative genomic studies, which showed that the corresponding gvp genes are present in all gas vesicle containing organisms examined [71,74-77]. …”

Gas Vesicles of Archaea and Bacteria

“…Cylindrical gas vesicles are also found in Halorubrum (Natronobacterium) vacuolatum isolated from the Kenyan alkaline soda lake Magadi, but they are only produced in media containing more than 15% salt (Fig. 1B) (Mwatha and Grant 1993;Mayr and Pfeifer 1997). The most unusual haloarchaeon is the large and square, but extremely thin gas-vesiculate Haloquadratum walsbyi (Fig.…”

“…Haloferax mediterranei harbors the related mc-vac region that leads to cylinder-shaped gas vesicles (Englert et al 1992a), whereas the Halorubrum vacuolatum cluster consists of the single nvgvpACNOFGHIJKLM transcription unit (Fig. 8;Mayr and Pfeifer 1997;Pfeifer 2004). The two regulator genes gvpDE have not been found so far.…”

“…A fourth gvp gene cluster (nv-vac) isolated from Halorubrum (Natronobacterium) vacuolatum harbours 12 gvp genes in a slightly different arrangement that are all transcribed as a single unit (Fig. 8) (Mayr and Pfeifer 1997;Pfeifer 2004). …”

Eight of Fourteen gvp Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea

“…Genes encoding proteins involved in gas vesicle formation have been identified in a few species of cyanobacteria (Anabaena flos-aquae, Calothrix strain PCC 7601) and in halophilic archaea (Halobacterium salinarum, Haloferax mediterranei, and the haloalkaliphilic archaeon Natronobacterium vacuolatum) (6,10,14,20,21,27), as well as in the soil bacterium Bacillus megaterium (26). In the case of halophilic archaea, 14 gvp genes cluster in an approximately 9-kb DNA region termed the vac region (10,11,19,20).…”