Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Correspondence
Dirk-Jan Scheffers
dirk-jan.scheffers{at}falw.vu.nl
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ABSTRACT |
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Present address: Molecular Microbiology, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
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INTRODUCTION |
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Recent work from various groups, focusing on the (re)distribution of several proteins during sporulation, has revealed intriguing examples of protein localization and distribution. At the start of sporulation, an asymmetric division septum is formed (a schematic overview of division and engulfment is shown in Fig. 1A). The switch from medial to asymmetric division is accomplished by an upshift in the expression of the key cell-division gene ftsZ, and a concomitant redistribution of FtsZ from a medial ring to a spiral that extends to both poles of the cell (Ben-Yehuda & Losick, 2002
). The spiral pattern is also observed for FtsA and EzrA (Ben-Yehuda & Losick, 2002
), which are other components of the division machinery that localize early in the division process (for a recent overview of B. subtilis cell division see Errington et al., 2003
). One of the two possible asymmetric division sites is then committed to division by the action of SpoIIE (Barák & Youngman, 1996
; Feucht et al., 1996
), resulting in the localization of other cell-division proteins to the asymmetric division site, and subsequent division (see Errington, 2003a
; Hilbert & Piggot, 2004
).
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Dynamic localization of proteins from the mother cell to the prespore has also been shown for the morphogenetic protein SpoIVA, which is involved in assembly of the spore cortex and coat (Lewis & Errington, 1996), and for SpoIVFB, a polytopic membrane protein involved in the activation of a transcription factor (Rudner et al., 2002
). In the latter case, Rudner et al. (2002)
showed that SpoIVFB is inserted into the cytoplasmic membrane in a dispersed fashion, after which the protein diffuses to, and is captured in, the outer prespore membrane (the diffusion-and-capture model).
This work is concerned with the localization of penicillin-binding proteins (PBPs), which are proteins involved in the synthesis of the cell wall PG during vegetative growth, cell division and sporulation (for recent reviews on cell wall synthesis during growth and sporulation, see Foster & Popham, 2001; Popham, 2002
) (Table 1
). Early work on expression profiles of PBPs during vegetative growth and sporulation indicated roles for PBPs 2B, 3, 4* and 5* in sporulation (Sowell & Buchanan, 1983
; Todd et al., 1983
). Studies on mutant strains, transcriptional profiling and localization have now identified a number of PBPs as playing (putative) roles during sporulation. The class A bifunctional transglycosylase/transpeptidase PBP1 is part of the division machinery that operates during asymmetric division, and is required for efficient sporulation (Scheffers & Errington, 2004
). Two other class A PBPs, 2c and 2d, play a redundant role in spore PG synthesis. A strain in which the genes for these PBPs 2c and 2d (pbpF and pbpG, respectively) are inactivated is incapable of completing sporulation, and shows defects in spore PG synthesis (McPherson et al., 2001
). The class B transpeptidase PBP2b, the only essential PBP in B. subtilis, is required for the asymmetric cell division, and localizes to the asymmetric septum (Daniel et al., 2000
). Another class B PBP, SpoVD, is essential for spore formation, and is required for the synthesis of cortical PG (Daniel et al., 1994
), whereas PBP4b does not seem to have an effect on spore PG, but is expressed under the control of the mother-cell-specific
E factor (Eichenberger et al., 2003
; Wei et al., 2004
). Finally, two low-molecular-mass PBPs are involved in spore PG synthesis. These are the carboxypeptidases 5* and DacF, which have partially redundant roles in regulating the degree of cross-linking of the spore PG, and a double mutant for both proteins has decreased spore heat resistance (Popham et al., 1995
, 1999
).
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METHODS |
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Construction of pbpX mutant strains.
Using primer pairs DJS149DJS150 and DJS151DJS152 (Table 3),
1·6 kb PCR fragments were generated containing the first 292 bp of pbpX plus upstream sequences, and the last 257 bp of pbpX plus downstream sequences. These fragments were cut with BamHI and EcoRI, respectively, and ligated to a BamHIEcoRI-digested PCR product containing a neo cassette, which was generated using primers km3 and km4, and plasmid pKM1 as a template. The ligation product was subjected to another PCR reaction using primers DJS149 and DJS152, and the resulting PCR product was transformed into strain 168, with selection for kanamycin resistance, generating strain 3906, which contains a deletion of pbpX codons 98305 (out of 391 codons). Correct integration of the ligation product into the chromosome was confirmed by PCR and sequencing.
Using primer pair DJS128DJS129, a fragment of pbpX (bp 161441) was amplified by PCR. The fragment was cut with HindIII and BamHI, and ligated into HindIIIBamHI-digested pMUTIN4, a vector that allows inactivation of the target gene as well as the monitoring of its expression through a transcriptional lacZ fusion (Vagner et al., 1998), generating pSG5313. pSG5313 was transformed into strain 168 to give strain 3905. Correct integration of the plasmid into the chromosome was confirmed by PCR.
Sporulation methods.
Sporulation was induced by growth to OD600 0·8 in CH, followed by resuspension in a starvation medium (SM; Partridge & Errington, 1993
; Sterlini & Mandelstam, 1969
). Cell pellets were washed with SM prior to resuspension to remove xylose, unless stated otherwise. Time zero (T0) was defined as the point at which the cells were resuspended in the starvation medium.
-Galactosidase activity was assayed as described by Errington (1986)
. One unit of
-galactosidase catalyses the production of 1 nmol 4-methylumbelliferone min1. Alkaline phosphatase activity was measured as described by Errington & Mandelstam (1983)
and Glenn & Mandelstam (1971)
. Sporulation efficiency was tested by determining the number of heat-resistant spores formed in the cultures at 10 h (T10) or 25 h (T25).
Microscopy.
Microscopy was performed essentially as described previously (Scheffers et al., 2004). Image acquisition was done as described by Lewis & Errington (1997)
, using Metamorph version 6.0 software (Universal Imaging Corporation). DNA was stained with Hoechst 33342 (1 µg ml1; Molecular Probes). Membranes were stained with FM95.5 (4 µg ml1; Molecular Probes). Images from a single focal plane were deconvolved using the No Neighbours algorithm from the Metamorph software package. Overlays of micrographs were assembled using Metamorph, before exporting the images to Adobe Photoshop version 6.0.
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RESULTS |
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When expressed during vegetative growth, GFPPBP2d localized in a dispersed fashion along the membrane, whereas GFPPBP4* localized in a punctate pattern (Fig. 2). These patterns were similar to the dispersed or punctate localization patterns observed with most PBPs expressed during vegetative growth (Scheffers et al., 2004
).
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A pbpX knockout strain has no distinguishable phenotype
Given the interesting localization pattern observed with GFPPbpX, I decided to study pbpX in more detail. pbpX was identified as a gene encoding an endopeptidase based upon sequence similarity (Foster & Popham, 2001), and it has recently been described as part of the
X regulon (Cao & Helmann, 2004
). pbpX was inactivated in two ways: by replacing 624 internal bases from the gene with a neo resistance marker, and by use of the pMUTIN-4 vector, which generates a lacZ transcriptional fusion to pbpX allowing the determination of the pbpX expression pattern (Methods). Both knockout strains grew at an identical rate, and with similar spore counts compared to wild-type B. subtilis (Table 4
). Correct formation of the asymmetric sporulation septum was followed by expression of
E-dependent genes, since activation of this sigma factor is dependent on septation (Piggot & Losick, 2001
). The
E-dependent synthesis of alkaline phosphatase was measured for both strains, and was found to be indistinguishable from wild-type (result not shown), showing that deletion of pbpX has no effect on septation during sporulation. The appearance of the
pbpX strain was indistinguishable from that of the wild-type (Table 4
). The transcriptional activation of pbpX followed a pattern typical for weak expression during vegetative growth, with no induction upon sporulation (result not shown).
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It is concluded that pbpX is a non-essential gene in B. subtilis, with no obvious phenotype during vegetative growth or sporulation.
PBP2c and PBP2d localize independently of each other
PBP2c and PBP2d play redundant roles during sporulation, and the presence of at least one of these PBPs is required for the synthesis of the spore germ cell wall (McPherson et al., 2001). The finding that both GFPPBP2c and GFPPBP2d localize to the prespore was in line with this observation. The localization of both GFP fusion proteins in the absence of the other PBP was studied. As shown in Fig. 5
, each of the proteins GFPPBP2c and GFPPBP2d was able to localize in the absence of the other protein. Again, for GFPPBP2d, xylose had to be present in the sporulation medium for continued synthesis. This shows that these proteins are not dependent on each other for correct localization, as might be expected from the fact that their functions appear to be redundant during sporulation (McPherson et al., 2001
).
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DISCUSSION |
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This paper is concerned with the localization of PG-synthesizing proteins during sporulation (summarized in Table 1). A collection of GFPPBP fusion proteins constructed earlier (Scheffers et al., 2004
) was used, in addition to new fusions to include PBPs implicated in sporulation. Expression of the GFPPBP fusion proteins is driven by the Pxyl promoter, which is switched on during vegetative growth, but switched off during sporulation by removal of xylose from the sporulation medium. This procedure reveals the localization of membrane proteins, and their redistribution upon sporulation, with no newly synthesized GFPPBPs in the prespore. Out of 11 GFPPBP fusions tested, only three showed a change in localization patterns during sporulation, and two of these fusions are to PBPs known to be involved in sporulation. This strongly suggests that the pattern changes are not caused by artefacts.
A striking change in protein localization was observed with GFPPbpX, which started by localizing to the division septum at midcell, and then appeared to spiral out in a pattern resembling FtsZ (Ben-Yehuda & Losick, 2002), and was then found at both asymmetric potential division sites. Although GFPPbpX spirals were rare, it should be noted that spiralling seems to be less obvious for membrane proteins than for cytosolic proteins (compare EzrAGFP to FtsZGFP and FtsAGFP in Ben-Yehuda & Losick, 2002
). GFPPbpX then appeared at both asymmetric septa, with unequal distribution of fluorescence intensity, as observed for SpoIIE by Wu et al. (1998)
. This observation adds credibility to the redistribution observed for GFPPbpX, since other cell-division proteins, notably PBPs (Daniel et al., 2000
; Scheffers & Errington, 2004
), only localize to the asymmetric septum when one of the potential division sites has been committed to division (see Hilbert & Piggot, 2004
). The unequal distribution possibly reflects which asymmetric division site is chosen for septum formation. This question could be resolved by following GFPPbpX distribution in individual sporulating cells with time. Despite this striking fluorescence pattern, pbpX does not play a critical role in B. subtilis. pbpX was inactivated in two ways, but effects on cell growth, cell shape or sporulation efficiency were not detected. Thus, PbpX cannot be an essential component of the cell-division machinery, but it could be associated with (a) component(s) from the division machinery, which it follows from the midcell division site to both asymmetric cell-division sites. A possible role for the endopeptidase PbpX is the quick removal of PG that connects two cells after vegetative division, or thinning of the sporulation septum prior to engulfment.
GFPPBP2c and GFPPBP2d, which are randomly distributed along the membrane in vegetative cells (Scheffers et al., 2004), are redistributed during sporulation: both proteins localized to the sporulation septum, followed the engulfing membrane, and were finally concentrated in the prespore membrane. There are two ways in which this redistribution can be achieved (see Fig. 1
). First, GFPPBP2c/d in the mother cell is recruited to the septum after septum closure, and then follows the mother cell membrane during engulfment. As a result, the majority of GFPPBP2c/d will be located in the outer prespore membrane (Fig. 1A
). Alternatively, GFPPBP2c/d can be recruited to the sporulation septum during septum formation, after which GFPPBP2c/d is found on both sides of the asymmetric septum. Following the membrane during engulfment results in GFPPBP2c/d being distributed throughout both inner and outer prespore membranes (Fig. 1B
). The fact that GFPPBP2c localizes to the engulfing membrane when expressed from the mother-cell-specific PspoIID, which is switched on only after closure of the sporulation septum, argues in favour of the first model.
PBP2c is expressed during both vegetative growth and sporulation, under control of G (Popham & Setlow, 1993a
). The observed redistribution presumably reflects the behaviour of the vegetatively expressed PBP2c during wild-type sporulation. Interestingly, since a pbpFpbpG double mutant has no detectable defects in its spore germ cell wall, which is synthesized from the surface of the inner prespore membrane, but is severely affected in its cortical PG, which is synthesized from the outer prespore membrane (McPherson et al., 2001
), it has been suggested that the more important site of PBP2c action is in the outer prespore membrane (Popham, 2002
). The observed pattern reflects this mode of action of PBP2c.
PBP2d expression is specific to the prespore (Pedersen et al., 2000), but when expressed as a GFP fusion protein during vegetative growth, GFPPBP2d localized in a dispersed fashion along the membrane, similar to various other PBPs described earlier (Scheffers et al., 2004
). To follow GFPPBP2d during sporulation, it was necessary to keep xylose present in the sporulation medium. Interestingly, even though GFPPBP2d was not expressed in the compartment in which it is expressed naturally, it did seem to recognize a targeting signal that guides it to the prespore septum and the engulfing membrane. Unfortunately, when expressed from sporulation-specific promoters, either in the prespore or in the mother cell, GFPPBP2d was degraded rapidly, making it impossible to confirm the localization of GFPPBP2d when expressed in the mother cell, or to study the localization of GFPPBP2d in the prespore in detail. PBP2c and PBP2d play redundant roles in sporulation (McPherson et al., 2001
), and in agreement with this, do not depend on each other for their localization to the prespore.
PBP2c and 2d show localization patterns that are similar to patterns observed for SpoIVFB, a protein that localizes to the prespore outer membrane by diffusion-and-capture (Rudner et al., 2002). We see two possibilities for the diffusion-and-capture of PBP2c and PBP2d. First, it is possible that PBP2c and PBP2d are actively targeted to the prespore, or captured at the prespore membrane, via an unidentified protein pathway. This active targeting could make sense for PBP2c, which is expressed in both mother cell and prespore, but not for PBP2d, which is expressed in the prespore alone in the wild-type situation. So, if this model were true, under our experimental conditions, GFPPBP2d redistribution should not be observed, unless the protein factor that is recognized is present in the space between inner and outer prespore membranes, and accessible to PBPs present in either membrane. Secondly, PBP2c and PBP2d may be recruited to the prespore by the presence of substrate or substrate analogues. GFPPBP2c and GFPPBP2d follow the engulfing membrane, which contains SpoIID, SpoIIM and SPoIIQ at its leading edge (Abanes-De Mello et al., 2002
). SpoIID is a cell wall hydrolase, which is suggested to use the cell wall as a track to drag along the membrane during engulfment (Abanes-De Mello et al., 2002
). The hydrolase activity of SpoIID would release PG building blocks that PBP2c and PBP2d could recognize as substrates, and maybe recycle by using them for synthesis of the spore germ wall or cortex, even before engulfment is complete. Recent work in Staphylococcus aureus (Pinho & Errington, 2005
) has shown that some high-molecular-mass PBPs depend on the presence of substrate for their correct localization. This has also been suggested for PBP localization in Streptococcus pneumoniae (Morlot et al., 2004
). Targeting of PBP2c and PBP2d to the prespore by the availability of substrate is an attractive model, although the question would remain of why there is a difference in substrate binding between PBPs 2c and 2d and the other high-molecular-mass PBPs.
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ACKNOWLEDGEMENTS |
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Received 11 October 2004;
revised 16 November 2004;
accepted 19 November 2004.
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