Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria

Abstract

SummaryDifferent modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, S erratia sp. ATCC 39006 (S39006). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006, identifying how this differs from the archaeon H alobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in E scherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E . coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain.

Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006

“…previously Tashiro et al, 2016). Briefly, for platebased assays, a normalized number of cells were spotted and dried onto LBA, and they were incubated at 30 C. The patch coloration, shape and opacity were assessed after 1, 2 or 5 days of growth.…”

“…These hollow cylindrical structures are permeable only to dissolved gas in the medium, and thus, gas vesicles reduce the density of bacterial cells, allowing them to fully colonize a static water column through flotation (Walsby, 1994). Gas vesicles are sensitive to pressure and production is often regulated in response to environmental and nutritional inputs from the organism's surroundings (Hechler & Pfeifer, 2009;Pfeifer et al, 2002;Ramsay et al, 2011;Tashiro et al, 2016). To date, all gas vesicles identified are composed of the small hydrophobic protein GvpA, which self-assembles into a ribbed array structure forming the gas vesicle walls (Buchholz et al, 1993;Hayes et al, 1988;and reviewed extensively in Walsby, 1994).…”

“…The genetic cluster contains three closely related isoforms of GvpA, designated GvpA1, GvpA2 and GvpA3, and three isoforms of GvpF, designated F1-F3. Recently, we determined that 11 of the 19 genes in the genetic locus were required for robust gas vesicle synthesis (Tashiro et al, 2016). That is, when any one of these genes was removed, either production of gas vesicles was abolished or, in the case of gvpV or gvpN, only small bicone gas vesicles were produced and these never developed into larger, cylindrical, mature forms.…”

“…That is, when any one of these genes was removed, either production of gas vesicles was abolished or, in the case of gvpV or gvpN, only small bicone gas vesicles were produced and these never developed into larger, cylindrical, mature forms. Additionally, in a gvpC mutant, while gas vesicles were produced, they were significantly less robust under pressure, suggesting that GvpC 39006 acts to strengthen gas vesicles, as it does in other systems (Tashiro et al, 2016). To demonstrate that, in each case, it was loss of the specific protein that prevented gas vesicle production, we expressed the WT genes from a plasmid in the cognate mutants to show restoration of gas vesicle production.…”

“…As we had previously demonstrated that plasmid-based expression of each essential gas vesicle protein was capable of complementation (Tashiro et al, 2016), we decided to examine further the impacts of overexpression of each protein essential for gas vesicle production in WT S39006.…”