Fig 1.
Visible stalk formation is rare among Brevundimonas species.
Brevundimonas species were cultured in PYE medium to mid-exponential growth phase, 10 images were taken of cells and the percentage of total cells bearing a visible stalk was calculated. This process was repeated three times and used to determine the average and standard deviation (% stalked). Between 454 and 3174 cells were counted for each species, with most species having over 1000 cells counted.
Fig 2.
Transmission electron microscopy of Brevundimonas species.
Brevundimonas species were cultured in PYE medium to mid-exponential growth phase and imaged by TEM. Cells were counted to determine the percentage of cells displaying polar extensions (% stalked). Between 92 and 248 cells were counted for each species. With some exceptions, percentages of stalked cells as determined by TEM were close to the number derived by phase contrast microscopy, supporting the results that these organisms produce stalks relatively rarely under nutrient rich conditions.
Fig 3.
Stalk formation is also variable in some Caulobacter species.
Caulobacter species were cultured in PYE medium to mid-exponential growth phase. A) 10 images were taken of cells and the percentage of total cells bearing a visible stalk was calculated. This process was repeated three times and used to determine the average and standard deviation (% stalked). Between 381 and 1138 cells were counted for each species. B) TEM imaging was performed on each strain and the percentage of cells displaying a polar extension was enumerated (% stalked). Between 83 and 151 cells were counted for each species.
Fig 4.
Deletion of pstS in B. subvibrioides leads to a growth defect and increased visible stalk biogenesis.
A) Growth curve of wild-type and ΔpstS B. subvibrioides strains in PYE media. Wild-type had a doubling time of 6.5 hrs while ΔpstS had a doubling time of 8.6 hrs. B) Incidence of stalk formation as a percentage of total cell population in wild-type and ΔpstS disruption strains for C. crescentus and B. subvibrioides. Disruption of pstS increased visible stalk incidence from 33.7 +/- 0.8% to 42.1 +/- 8.8% in C. crescentus, and from 1.6 +/- 0.6% to 24.5 +/- 11.0% in B. subvibrioides.
Fig 5.
Incidence of stalk and/or holdfast detection in wild-type and ΔpstS B. subvibrioides.
Wild-type and ΔpstS strains of B. subvibrioides were cultured in triplicate and labeled with fluorescently-conjugated Wheat Germ Agglutinin lectin, followed by phase contrast and epifluorescence microscopy. Cells were scored on whether they had a detectable stalk, holdfast, both, or neither. A) Percentage of the total population with a detectable holdfast. B) Percentage of non-stalked or stalked cells with a detectable holdfast. C) Percentage of total population that had no visible polar structure (i.e. no stalk or holdfast), stalk, holdfast, or both. Deletion of pstS led to an increase in stalk incidence, but the majority of stalked cells had no detectable holdfast.
Fig 6.
Holdfast was detected at the tip of stalks in wild-type and ΔpstS B. subvibrioides stalked cells.
Staining of polysaccharide by fluorescently-conjugated wheat germ agglutinin lectin shows that holdfast material is located at the tip of the stalk when stalks are formed by B. subvibrioides wild-type or ΔpstS cells. Stained cells were imaged by phase contrast (Ph) and epifluorescence (Fl) microscopy, and images were overlaid (O). In some instances, several cells were joined at the holdfast forming a “rosette”, a common occurrence in C. crescentus.
Fig 7.
Time-lapse microscopy of stalked wild-type B. subvibrioides cells under nutrient rich conditions.
Wild-type B. subvibrioides cultures were grown to mid-exponential phase in rich growth medium, and then mounted on rich medium agarose pads. The growth of 20 stalked cells was observed every 30 minutes for 8 hrs, typically covering two cell divisions. A) A representative time-lapse is presented, demonstrating two stalked cells growing and dividing, both producing non-stalked cells as well as seemingly losing their stalks. B) Selected images from time-lapse experiments showing B. subvibrioides cell potentially losing stalks.
Fig 8.
Time-lapse microscopy of C. crescentus cells shifted from nutrient-poor to nutrient-rich conditions.
Wild-type C. crescentus was cultured in phosphate-limited HIGG growth media to mid-exponential phase to induce longer than usual stalks, then cells were mounted on rich medium agarose pads. The growth of 20 stalked cells was observed every 30 minutes for 8 hrs. A) A representative time-lapse is presented where a cell with a very long stalk progresses through multiple divisions while appearing to maintain stalk length. B) Selected images from time-lapse experiments showing cells with long stalks maintaining stalk length.
Table 1.
Conservation of stalk-related genes in members of the Caulobacterales clade1.
Fig 9.
Soluble GFP is not detected in B. subvibrioides stalks.
The plasmid pRVGFPC-2 was moved into wild-type and ΔpstS strains; upon vanillate induction soluble GFP is produced. This construct was moved into wild-type and ΔpstS B. subvibrioides strains. A) Cells were imaged in the absence (- van) or presence (+ van) of vanillate. Imaging was performed by phase contrast (Ph) and epifluorescence (Fl) microscopy, and the images were overlaid (O). Upon examination, cytoplasmic GFP does not enter the stalk compartment. B) Additional examples of ΔpstS cells with soluble GFP absent from stalks.
Fig 10.
PstA-GFP is detectable in some B. subvibrioides stalks.
The B. subvibrioides pstA gene was cloned into plasmid pRVGFPC-2, a replicating plasmid that creates a C-terminal GFP fusion as well as place the gene under a vanillate inducible promoter. This construct was moved into wild-type and ΔpstS B. subvibrioides strains. A) Cells were imaged in the absence (- van) or presence (+ van) of vanillate. Imaging was performed by phase contrast (Ph) and epifluorescence (Fl) microscopy, and the images were overlaid (O). PstA-GFP is detectable in some stalk compartments. B) More examples of PstA-GFP imaging. Top left and right: examples of fluorescence detected in stalks. Bottom left: examples of fluorescence not detected in stalks. Bottom right: example of fluorescence detected in a portion of a stalk.
Fig 11.
B. subvibrioides prevents soluble GFP from entering the stalk but allows StpX into the stalk.
The C. crescentus stpX gene was cloned into plasmid pRVGFPC-2, a replicating plasmid that creates a C-terminal GFP fusion as well as place the gene under a vanillate inducible promoter. This construct was moved into wild-type and ΔpstS B. subvibrioides strains. A) Cells were imaged in the absence (- van) or presence (+ van) of vanillate. Imaging was performed by phase contrast (Ph) and epifluorescence (Fl) microscopy, and the images were overlaid (O). Upon examination, fluorescence was detected in both the stalk and cell body. B) Additional examples of fluorescence in stalks and cell body.