Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli

Abstract

Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E. coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-contrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In B. megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E. coli. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.

FIG. 1

The gvp gene cluster and contiguous sequences in B. megaterium B001S. (A [from top to bottom]) Tn917-LTV1 and contiguous sequences in the chromosome of strain B001S and relevant restriction enzyme sites; cloned fragments of DNA in pNL4, pNL21, and pNL22 contiguous with the IR-L sequences; cloned DNA fragment in pNL24, map of putative genes, intergenic regions in base pairs (igrs), and relevant restriction enzyme sites; and ruler of sequence in base pairs. (B) Subclones of pNL24 and their ability to produce gas vesicles in E. coli. Gas vesicles were observed as refractile bodies by phase-contrast microscopy. ++++++, gas vesicles throughout all cells in culture; +++++, slightly reduced quantity of gas vesicles in most cells; +++, gas vesicles in approximately 50% of cells and in reduced quantity; +, very few gas vesicles in about 10% of cells; −, no gas vesicles observed. IR-L and IR-R, inverted repeats at lacZ end (left) and Tn917 end (right), respectively; ColE1, E. coli origin of replication; lacZ, β-galactosidase gene; cat, chloramphenicol resistance gene; bla, ampicillin resistance gene; erm, erythromycin resistance gene.

FIG. 2

Homologies of predicted amino acid sequences. (A) Multiple sequence alignment of B. megaterium GvpA and GvpB with representative homologs from cyanobacteria and the halophilic archaea, with the majority consensus sequences shown in black. (B) Multiple sequence alignment of B. megaterium GvpA, GvpB, GvpJ, GvpS, and GvpK, with multiple consensus sequences shown in black. (C) Pairwise alignment of H. halobium GvpI with B. megaterium GvpQ, with amino acid identities shown in black. The Clustal method with the PAM250 residue weight table was used. GVPA_APHFL, GvpA of A. aquae-flos; GvpA1_HALHA, GvpA (plasmid borne) of H. halobium; GVPI_HALHA, GvpI of H. halobium; Bmeg, B. megaterium.

FIG. 3

Buoyancy of E. coli strains carrying plasmids with cloned B. megaterium gvp genes as labeled: pNL25, pNL26, pNL29, and the control, pKS (pBluescriptIIKS).

FIG. 4

DIC microscopy of E. coli(pNL26) (A) and E. coli(pBluescriptIIKS) (B). In panel A, the cells are longer and have hollow-looking structures (arrow), and most of the cells tend towards a vertical position in the wet mount compared to those in panel B. Magnification, ×1,000.

FIG. 5

Pressure sensitivity of refractile bodies in E. coli(pNL26). See Materials and Methods for details. Triplicate samples of the same culture had a 5% margin of error.

FIG. 6

Electron micrographs of E. coli(pNL26). (A) Protoplast of a dividing cell shows gas vesicles within the cell. Bar, 123 nm. (B) Gas vesicles from cell lysate. Bar, 44 nm.