Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus

Abstract

The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.

Fig 1

Accumulation of PBP2 at the FtsZ ring location. (A) JAT878 cells producing mCherry-PBP2 under the control of the vanillate-inducible promoter on the chromosome were grown in the presence of vanillic acid for 3 h in PYE medium before imaging on an M2G pad. (B) CJW2135 cells producing GFP-PBP2 under the control of the xylose-inducible promoter on the chromosome were grown in PYE with xylose for 3 h before imaging on an M2G pad. (C) CJW2742 cells producing GFP-PBP2 from the native promoter on the chromosome in place of wild-type PBP2 were grown in PYE and imaged on an M2G pad. Bar, 1 μm. (D) CJW3402 cells were grown in PYE with xylose for 2 h before synchronization. Synchronized swarmer cells were resuspended in PYE medium containing xylose, and samples were taken every 20 min for microscopy on M2G pads. Bar, 1 μm.

Fig 2

PBP2 colocalizes with MreB near midcell in an FtsZ-dependent but MreB-independent manner. (A) Demographs of CJW3402 cells producing FtsZ-YFP and CFP-PBP2. Cells were grown in PYE, and the synthesis of FtsZ-YFP and CFP-PBP2 was induced by adding xylose and vanillic acid for 4 h prior to imaging on an M2G pad. (B) Micrographs showing the localization of GFP-PBP2 in FtsZ-depleted cells. CJW3396 cells were grown in PYE containing vanillic acid, the inducer of ftsZ expression. Xylose, the inducer of gfp-pbp2 expression, was added to the culture 2 h prior to cell synchronization. Swarmer cells were isolated and resuspended in PYE containing xylose but not vanillic acid. After 2 h of growth and FtsZ depletion, the cells were spotted and imaged on M2G pads. (C) Demographs and representative micrographs of cells (strain CJW3397) producing YFP-MreB and CFP-PBP2. Cells were first grown in PYE medium with xylose and vanillic acid for 4 h to induce the expression of YFP-MreB and CFP-PBP2, respectively, prior to microscopy on M2G agarose pads. (D) Fluorescent micrograph showing A22-treated cells producing GFP-PBP2 (strain CJW2135). Cells were grown in PYE medium containing xylose for 2 h to induce GFP-PBP2 synthesis followed by 2 h of A22 (50 μM) treatment before microscopy on M2G pads. (E) Micrograph showing the localization of GFP-PBP2 in CJW3403 cells carrying the mreB_Q26P mutation. Cells were grown in PYE, and synthesis of GFP-PBP2 was achieved by addition of xylose for 4 h prior to imaging on M2G pads. Bar, 1 μm.

Fig 3

Relocation of PBP2 to the division site is dependent on changing the growth medium from PYE to M2G. Demographs and representative images of JAT878 cells producing mCherry-PBP2 after 3 h of induction with vanillic acid. (A) Cells from a PYE culture were imaged on an M2G pad. (B) Cells from an M2G culture were imaged on an M2G pad. (C) Cells from a PYE culture were imaged on a PYE pad. (D) Cells from an M2G culture were imaged on a PYE pad. Bars, 1 μm.

Fig 4

Relocation of PBP2 to midcell happens fast upon medium change whereas the return of PBP2 to its dispersed localization is slow and growth dependent. (A) Demographs showing mCherry-PBP2 localization at selected time points (40 s, 2 min, and 5 min) following an osmotic upshift. JAT878 cells producing mCherry-PBP2 (by induction with vanillic acid for 3 h) were grown in PYE and imaged on an M2G pad lacking vanillic acid. The first image was acquired 40 s after spotting cells on the pad. Additional images were taken every 20 s for up to 5 min. (B) Demographs showing mCherry-PBP2 localization following a brief wash in either M2G (osmotic upshift) or PYE (no osmotic shock). JAT878 cells producing mCherry-PBP2 (by induction with vanillic acid for 3 h) were grown in PYE and then washed in M2G or PYE medium before being spotted on a PYE pad. (C) Demographs showing mCherry-PBP2 localization at selected time points following an osmotic upshift. JAT878 cells producing mCherry-PBP2 (by induction with vanillic acid for 3 h) were grown in PYE and spotted on M2G for time-lapse imaging. (D) Same procedure as that in panel C except that cells were washed once with M2 (containing all the constituents of M2G except glucose) and spotted on an agarose pad containing M2.

Fig 5

An upshift in medium osmolality is sufficient to cause PBP2 recruitment to the FtsZ ring location. (A) The graph shows average osmolality measurements of indicated solutions obtained from 3 independent measurements. Error bars indicate the standard deviations of measurement. (B) Demographs showing mCherry-PBP2 localization from PYE cultures of JAT878 cells that have been briefly washed in solutions of different osmolalities prior to imaging on PYE pads. mCherry-PBP2 synthesis was achieved by addition of vanillic acid for 3 to 4 h in the PYE cultures. (C) Same procedure as that in panel B except that M2G cultures and M2G pads were used.

Fig 6

C. crescentus growth is very sensitive to external osmolality. (A) The graph shows average osmolality measurements of indicated solutions obtained from 3 independent measurements. Error bars indicate the standard deviations of measurement. (B) Wild-type CB15N cells were grown in 3 replicates in either M2G or M2G_half (which consists of half the concentrations of glucose, phosphate buffer, and nitrate found in M2G) supplemented with 0, 20, 40, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM NaCl. Growth was monitored using a microplate reader, and doubling times in the exponential phase of the cultures were plotted as a function of NaCl concentration (top) or osmolality of the growth medium (bottom). The plots show mean values ± SDs.

Fig 7

The localization of E. coli PBP2 is largely unaffected by osmotic upshifts. Demographs showing GFP-PBP2 localization under different growth conditions. In all cases, E. coli cells (strain LMC1840) were grown at 30°C and induction of GFP-PBP2 synthesis was achieved by addition of IPTG for 40 min. (A) Cells were grown in LB medium and imaged on pads containing either LB or LB plus 100 mM NaCl. (B) Cells were grown in M9G medium and imaged on pads containing either M9G or M9G plus 100 mM NaCl. (C) Cells were grown in PYE medium and imaged on pads containing either PYE or PYE supplemented with the indicated concentration of NaCl.

Fig 8

The localizations of PBP1a and RodA are also affected by osmotic upshifts in C. crescentus. (A) Demographs and representative images showing YFP-RodA localization in C. crescentus cells (strain CJW3207) grown in PYE medium and imaged on pads containing either M2G (no shift) or PYE (upshift). YFP-RodA synthesis was achieved by addition of xylose for 3 h before spotting cells on an agarose pad. Arrowheads indicate accumulation of YFP-RodA near midcell. Bars, 1 μm. (B) Same procedure as that in panel A except that CJW3288 cells producing GFP-PBP1a were imaged. Synthesis of GFP-PBP1a was induced with xylose 3 h prior to imaging. (C) Demographs and representative images showing the localization of YFP-RodA and mCherry-PBP2. CJW4621 cells were grown in PYE with xylose and vanillic acid for 3 h to induce the synthesis of both YFP-RodA and mCherry-PBP2 prior to imaging on an M2G pad. Bars, 1 μm. (D) Same procedure as that in panel C except that CJW4620 cells producing GFP-PBP1a and mCherry-PBP2 were imaged following induction of their synthesis with xylose and vanillic acid for 3 h.