In eubacteria, the cell wall is the principal shape-maintaining and stress-bearing element of the cell, functioning as the bacterial ‘exoskeleton’ (for reviews, see Höltje, 1998; Foster and Popham, 2001). Therefore, bacterial morphogenesis is closely related to growth of the cell wall. Until recently, the growth and shape of rod-shaped bacteria such as Bacillus subtilis was thought to be mediated by a passive process of lateral insertion of cell wall material during elongation, and transverse insertion of septal cell wall material during septation. The recent discovery of an actin-like cytoskeleton in B. subtilis (Jones et al., 2001) has altered this view. Two proteins, MreB and Mbl, form filamentous, helical structures just below the cytoplasmic membrane, which are made up of polymers that show a striking resemblance to actin polymers (van den Ent et al., 2001). These structures determine the shape of the cell, and the occurrence of at least one mreB-like gene is closely linked to bacteria that have a complex (i.e. non-coccoid) shape (Jones et al., 2001). Recent work from our laboratory has shown that Mbl governs the insertion of new cell wall material in a helical pattern in B. subtilis (Daniel and Errington, 2003). However, how the connection between the internal actin-like cytoskeleton and the exoskeleton formed by the cell wall is made remains elusive.
The cell wall of B. subtilis consists of peptidoglycan (PG), glycan strands with peptide side-chains that are highly cross-linked, and covalently linked anionic polymers (Foster and Popham, 2001). The polymerization and cross-linking of peptidoglycan is mediated by penicillin-binding proteins (PBPs). The PBPs can be subdivided into three classes (see Table 1): class A high-molecular-weight (high-MW) PBPs, with both a transglycosylase and a transpeptidase domain; class B high-MW PBPs, with an N-terminal domain of unknown function and a transpeptidase domain; and a class of low-molecular-weight (low-MW) PBPs with d,d-carboxypeptidase or endopeptidase activity (Goffin and Ghuysen, 1998; Foster and Popham, 2001). B. subtilis PBPs function in both vegetative growth and the formation of the primordial cell wall and cortex of endospores. Strikingly, both B. subtilis and Escherichia coli contain large numbers of genes coding for PBPs, 16 and 12, respectively, of which products have been detected biochemically for 11 (B. subtilis) and 12 (E. coli ) (Foster and Popham, 2001). Studies in B. subtilis (Popham and Setlow, 1996; Popham et al., 1999; McPherson et al., 2001; McPherson and Popham, 2003), E. coli (Denome et al., 1999) and Streptococcus pneumoniae (Hoskins et al., 1999; Paik et al., 1999) of single and multiple inactivations of pbp genes suggest a redundancy in function for both high-MW and low-MW class PBPs.
|Gene||Protein||Expression||Localization during vegetative growth|
|Class A PBPs (bifunctional transglycosylase/transpeptidase)|
|ponA||PBP1a/b||vega,1||Septal2, septal, low fluorescence at periphery, this work|
|pbpF||PBP2c||veg, late stages of spor3||Septal, low fluorescence at peripheryb|
|pbpD||PBP4||veg4||Septal, distinct spots at peripheryb|
|Class B PBPs (transpeptidase)|
|pbpA||PBP2a||veg6||Evenly distributed along the membrane|
|pbpB||PBP2b||veg, spor7||Septal8, this work|
|pbpC||PBP3||veg, low expression during spor9||Distinct foci and bands at cell periphery|
|pbpH||PbpH||veg11||Evenly distributed along the membrane|
|yrrR||YrrR||Not known||Evenly distributed along the membrane|
|Low-MW PBPs (carboxypeptidase)|
|dacA||PBP5||Not known||Septal, distinct spots at periphery|
|dacC||PBP4a||Late stationary phase13||Distinct foci and bands at cell periphery|
|Low-MW PBPs (endopeptidase)|
|pbpX||PbpX||Not known||Septal, low fluorescence at periphery|
Previous studies on PG synthesis in E. coli and B. subtilis revealed the dispersed insertion of cell wall material along the cylindrical part of the cell during vegetative growth (Burman et al., 1983; Mobley et al., 1984; Schlaeppi et al., 1985; Woldringh et al., 1987; de Pedro et al., 1997). Biochemical evidence from B. subtilis, E. coli and Haemophilus influenzae points to cell wall synthesis being mediated by a ‘holoenzyme’ composed of murein synthases and hydrolases that track along the peptidoglycan to insert new PG strands into the cell wall (Bhardwaj and Day, 1997; Höltje, 1998; Alaedini and Day, 1999; Simon and Day, 2000). Several PBPs would form part of such a holoenzyme, providing transglycosylase, transpeptidase, carboxypeptidase and endopeptidase activities, together with hydrolases that remove old glycan strands from the cell wall. Given the difference in cell wall synthesis between elongation and division, specific holoenzyme complexes for both processes have been proposed (Höltje, 1998). One difference between the holoenzyme involved in cell wall elongation or invagination during division would be the presence of a cell division-specific transpeptidase. Two multienzyme complexes with different PBP composition have indeed been found in H. influenzae, and one complex contained PBP2, implicated in elongation, whereas the other contained PBP3, implicated in division (Alaedini and Day, 1999). In both B. subtilis (PBP2b; Daniel et al., 2000) and E. coli (PBP3; Weiss et al., 1997), the cell division-specific transpeptidase is localized at the septum. As E. coli only contains one other class B high-MW transpeptidase, PBP2, this was proposed to be a unique component of the elongation complex (Höltje, 1998). Recently, E. coli PBP2 was shown to localize in a spot-like pattern in the lateral cell wall as well as to the septum in dividing cells (Den Blaauwen et al., 2003), showing that this PBP does not localize exclusively to the lateral wall. B. subtilis PBP1 localizes to the cell division septum as well (Pedersen et al., 1999). These are the only PBPs for which localization has been studied. In B. subtilis, PBPs that localize to the lateral cell wall have not yet been found. Two candidates for lateral wall localization in B. subtilis are PBP2a and PbpH (Murray et al., 1998; Wei et al., 2003). Single knock-outs of these genes are indistinguishable from the wild type, but a double mutant is non-viable, and cells of a pbpA mutant expressing pbpH from an inducible promoter became round before lysing, after depletion of the inducer (Wei et al., 2003). Also, germinating, ovoid-shaped, spores lacking PBP2a require more time to attain the cylindrical shape characteristic of vegetative cells (Murray et al., 1998).
We wanted to study the localization of PBPs in B. subtilis to determine where peptidoglycan synthesis occurs and to search for a link between the sites of peptidoglycan synthesis and active cell shape determination by the MreB/Mbl cytoskeleton. N-terminal green fluorescent protein (GFP) fusions to all the vegetatively expressed PBPs of B. subtilis were constructed, and their localization was determined by fluorescence microscopy. The data show that PBPs localize in several distinct patterns. Some PBPs localize specifically to the lateral part of the cell, whereas others are localized specifically to the septum. Other PBPs show localization to both the septum and the lateral wall, sometimes with an irregular distribution of fluorescence. The patterns of GFP–PBP localization are not markedly altered in the absence of either MreB or Mbl. The results show that PBPs localize in an organized rather than a dispersed fashion and implicate PBP3, PBP5, PBP4a, and possibly PBP4, in lateral cell wall synthesis.