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. 2011 Oct 11;2(5):e00202-11.
doi: 10.1128/mBio.00202-11. Print 2011.

Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division

Ho-Ching Tiffany Tsui  1 , Susan K Keen, Lok-To Sham, Kyle J Wayne, Malcolm E Winkler
Affiliations

"V体育官网入口" Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division

Ho-Ching Tiffany Tsui et al. mBio. .

Abstract (V体育ios版)

The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed "ExPortal," in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥ 75%) of exponentially growing cells, S. pneumoniae SecA (SecA (Spn)) and SecY (Spn) located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA(Spn) and SecY(Spn) remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells VSports手机版. In contrast, the HtrA (Spn) homologue localized to the equators and septa of most (> 90%) dividing cells, whereas the SrtA(Spn) sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. .

Importance: Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrA(Spn)) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecA(Spn), SecY (Spn), and HtrA (Spn) to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrA(Spn) may play a general role in quality control of proteins exported by the Sec translocase V体育安卓版. .

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FIG 1
FIG 1
SecA-L-FLAG3 localizes primarily to equators, septa, and hemispheres of exponentially growing S. pneumoniae cells. Strain IU4472 (D39 ∆cps rpsL1 secA-L-FLAG3) was grown to mid-exponential phase in BHI broth, and DAPI staining and IFM of cells were performed as described in Materials and Methods. Cells at different stages of division were binned based on images from phase-contrast microscopy (column 1), and localization patterns of DAPI (pseudocolored red; column 2) and SecA-L-FLAG3 (pseudocolored green; column 3) were determined from images obtained by epifluorescent microscopy. Columns 4 and 5 show overlays of columns 1 and 3 or columns 1 and 2, respectively. Images of representative cells are shown at different stages of division. A schematic summary at right shows the SecASpn localization pattern for >75% of the cell population at each stage (Fig. 2). The experiment was performed more than 5 times independently. Multiple fields were observed in each experiment, with similar results. Scale bar = 1 µm.
FIG 2
FIG 2
Distribution of predivisional, early divisional, and late-divisional cells with SecA-L-FLAG3 (A) and HtrA-L-FLAG3 (B) localized at equators, division septa, or hemispheres between septa and future equators, as indicated. Cells were grown exponentially (log) or to the transition to stationary phase, and IFM was performed as described in Materials and Methods. (A) cls+ parent strain IU4472 (D39 ∆cps rpsL1 secA-L-FLAG3), ∆cls::Pc-erm mutant IU5093, and complemented ∆cls mutant IU5138 (Δcls::Pc-erm//CEP::cls+, where “//” indicates a separate ectopic location in the chromosome). (B) cls+ parent strain IU4468 (D39 ∆cps rpsL1 htrA-L-FLAG3), ∆cls::Pc-erm mutant IU5089, and complemented ∆cls mutant IU5136 (∆cls::Pc-erm/CEP::cls+). See the text for additional details.
FIG 3
FIG 3
SecA-L-FLAG3 (A) and HtrA-L-FLAG3 (B) do not localize in cells during the transition from exponential to stationary phase in BHI broth. Strains IU4472 (Fig. 1) and IU4468 (Fig. 4) were grown to transition phase, and DAPI staining and IFM of cells were performed as described in Materials and Methods. Representative cells are shown with the same column arrangement as in Fig. 1. The experiment was performed more than 2 times independently. Multiple fields were observed in each experiment, with similar results. Scale bar = 1 µm.
FIG 4
FIG 4
HtrA-L-FLAG3 localizes to equators and septa of exponentially growing S. pneumoniae cells. Strain IU4468 (D39 ∆cps rpsL1 htrA-L-FLAG3) was grown to mid-exponential phase in BHI broth, and DAPI staining and IFM of cells were performed as described in Materials and Methods. Representative images are shown with the same row and column arrangements as in Fig. 1. A schematic summary at right shows the HtrASpn distribution pattern in >90% of the cell population (Fig. 2). The experiment was performed more than 3 times independently. Multiple fields were observed in each experiment, with similar results. Scale bar = 1 µm.
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References

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