Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain1
Max-Planck-Institut für Entwitcklungsbiologie, Spemannstrasse 35, D-72076 Tübingen, Germany2
Author for correspondence: Miguel Angel de Pedro. Tel: +34 91 3978083. Fax: +34 91 3978087. e-mail: madepedro{at}cbm.uam.es
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ABSTRACT |
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Keywords: ftsZ, pbpB, penicillin-binding protein, beta-lactam, murein
Abbreviations: PBP, Penicillin-binding protein; SMS, septal murein synthesis
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INTRODUCTION |
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In Escherichia coli the mode of insertion of new precursors into the sacculus apparently shifts from diffuse to zonal at the initiation of septation (de Pedro et al., 1997 ). Lateral wall elongation is accomplished by intercalation of new and pre-existing murein all over the cylindrical surface of the sacculus. At the time of septation an area of highly localized murein synthesis develops into the potential division site and remains active until septum completion (SMS, septal murein synthesis). Once the dividing sacculus is split, SMS stops and the new cell wall poles become metabolically inert. Activation of SMS requires the activity of FtsZ, but not the essential division proteins FtsA, FtsQ and PBP3 (penicillin-binding protein 3). Inactivation of FtsZ leads to the generation of filamentous cells in which murein precursors are inserted in a diffuse way for as long as the filament elongates. In contrast, cells from ftsA, ftsQ or ftsI mutants growing at the restrictive temperature are able to trigger SMS in due time. However, SMS remains active only for a defined period of time. Then it is switched off and murein synthesis reverts to a diffuse insertion mode. When the time for the next round of septation is reached and FtsZ assembles into new Z-rings, SMS is triggered again and the whole process repeats. The consequences are that diffuse and zonal modes of murein synthesis alternate and that filaments of ftsA, ftsQ or ftsI, but not ftsZ, mutants develop rings of all new murein at the potential division sites (de Pedro et al., 1997
).
Growth of the bacterial cell sacculus is thought to require the concerted action of biosynthetic and hydrolytic enzymes (Höltje, 1996 , 1998
; Höltje & Heidrich, 2001
; Weidel & Pelzer, 1964
). The former are essential enzymes which mediate elongation of nascent peptidoglycan strands as well as their insertion into the sacculus. An essential role for murein hydrolases has been repeatedly postulated on theoretical grounds. As the sacculus is a covalently closed structure, its enlargement requires cleavage of pre-existing covalent bonds (Höltje, 1998
; Shockman & Höltje, 1994
; Weidel & Pelzer, 1964
). Nevertheless, none of the E. coli murein hydrolases known seems to be essential for enlargement. Even multiple mutants are capable of essentially normal growth in length (Höltje & Heidrich, 2001
; Shockman & Höltje, 1994
). In contrast, a critical requirement of murein hydrolases for cell division has been substantiated (Blackman et al., 1998
; Heidrich et al. 2001
; Höltje & Heidrich, 2001
; Tomasz, 1974
). In E. coli the activity of amidases, acting in concert with endopeptidases and lytic translgycosylases, is necessary for proper septum splitting (Heidrich el al. 2001
; Höltje & Heidrich, 2001
). This situation has led to the recent proposal of a model in which only septation would require the concerted action of synthetases and hydrolases. Enlargement of the sacculus would be mediated by murein transferases (Höltje & Heidrich, 2001
). It is speculated that enzymes exist that catalyse a transpeptidation between old and nascent murein by using the chemical energy present in pre-existing peptide cross-bridges. Such enzymes would cleave bonds in the sacculus concomitantly with the insertion of new material, therefore obviating the need for additional hydrolases.
Induction of bacterial lysis by ß-lactams and other murein synthesis inhibitors normally requires active growth of the cells and the activity of murein hydrolases (autolysins) (Tomasz, 1974 ; Tuomanen & Tomasz, 1990
; Leduc & van Heijenoort, 1980
; Leduc et al., 1982
). These observations have led to models proposing that lysis occurs because the inhibitors break the balance between biosynthetic and hydrolytic enzymes, favouring uncontrolled murein degradation by the latter (Koch, 2000
; Rogers et al., 1980
; Weidel & Pelzer, 1964
). However, the lack of hard evidence in support of the implication of hydrolases in elongation of the sacculus and the demonstration of ß-lactam-induced lysis in non-growing cells under specific conditions (Ishiguro & Kusser, 1988
; Kusser & Ishiguro, 1986
, 1987
) has led to alternative models in which lysis is the consequence of the activation of lytic enzymes which under normal growth conditions would be partially or totally inhibited (Ishiguro & Kusser, 1988
; Tomasz, 1983
, 1984
).
Early observations of ß-lactam-induced bacteriolysis showed the preferential occurrence of lysis at potential division sites (Staugaard et al., 1976 ; Tomasz, 1974
), which, in the case of E. coli, often resulted in sacculi with a sharp cut at the corresponding location (Schwarz et al., 1969
). These observations suggest that SMS might be particularly prone to provoke lysis when disturbed. The zonal nature of SMS suggests that the local concentration of biosynthetic complexes might be higher at division sites than anywhere else in the cell. If biosynthetic complexes require the concomitant activity of hydrolases, damage inflicted by bacteriolytic agents should be more pronounced at the places with higher local densities. Furthermore, as septation itself needs the activity of murein hydrolases to split nascent septa (Heidrich et al. 2001
; Höltje & Heidrich, 2001
), bacteriolytic agents could trigger lysis more effectively at those areas where hydrolases are actually performing their function and loss of control would have immediate consequences. According to such ideas, the mode of lysis of filament cells should be strongly dependent on whether or not SMS is activated during growth under restrictive conditions. In the former case lysis should occur as sharp cuts at the position of the rings of all new murein generated by the activation of SMS. In the latter, as no regions of localized synthesis develop, a general weakening of the sacculus network, with essentially random break points, is more likely to be observed. Examination of cefsulodin-induced lysis in sacculi from filament cells of E. coli ftsZ and ftsI mutants confirmed these predictions.
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METHODS |
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Purification of sacculi.
Culture samples (ca 1010 cells) were harvested by centrifugation (10000 g, 3 min) at the growth temperature. The pellets were quickly resuspended into 3 ml 0·9 g NaCl l-1, transferred into test tubes containing 6 ml 8% (w/v) SDS and kept closed on a boiling water bath for 6 h with magnetic stirring. Sacculi were further processed for electron microscopy as described by de Pedro et al. (1997) .
Electron microscopy of sacculi.
Carbon-pioloform-coated copper grids (200# mesh) were glow-discharged (10 min) and floated for 15 min on drops of sacculi suspensions appropriately diluted in double-distilled water. Grids were then removed, excess liquid was absorbed on filter paper and grids were allowed to air dry for 10 min. Grids were washed (five times) by flotation on distilled water drops, stained by floating for 1 min on 1% (w/v) uranyl acetate in water, briefly washed in water and air-dried. To better visualize the flatness of sacculi, grids were in some instances subjected to carbon/platinum shadowing at an angle of 15°. Microscopic observations were performed on a Philips CM10 transmission electron microscope at an acceleration voltage of 60 kV.
Muramidase digestion of murein sacculi.
On the grid digestion of sacculi was performed as described by de Pedro et al. (1997) . In short, purified sacculi were deposited on electron microscope copper grids and washed as described above. Grids were floated for 1 min on drops (50 µl) of a solution of Cellosyl muramidase (Aventis) at 5 µg ml-1 in 20 mM sodium phosphate buffer, pH 4·9, at room temperature. To stop the reaction the enzyme solution was removed from the grid with filter paper, grids were rapidly washed three times in drops of precooled (46 °C) double-distilled water and stained for 1 min by flotation on drops of 1% (w/v) uranyl acetate in water.
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RESULTS |
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DISCUSSION |
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The periodic alternating use of diffuse and zonal modes of murein growth helps to explain the old observation that ß-lactams apparently induce lysis in accordance with cell division in normally dividing cells. Induction of lysis by ß-lactams proceeds at a rate related to the growth rate and very often cells blow up at positions corresponding to division sites (Leduc & van Heijenoort, 1980 ; Leduc et al., 1982
; Schwarz et al., 1969
; Staugaard et al., 1976
). According to our interpretation in any cell able to trigger SMS, lysis should occur preferentially at the division site. As generation of division sites is intimately related to growth rate, the rate of lysis should be automatically linked to the rate of growth.
The link between induction of fast, localized cell lysis and activation of SMS at division sites could explain previous, and apparently contradictory, results about the lytic response to cefsulodin of synchronized cultures of E. coli. In experiments performed with cells synchronized by thymine/amino acid starvation, addition of cefsulodin at any time prior to termination of DNA replication resulted in cell lysis at the time of division, but not earlier (Garcia del Portillo et al., 1989 ). In contrast, in similar experiments performed with cells synchronized either by the Baby Cell Machine (membrane elution) (Jacoby & Young, 1991
) or centrifugal elutriation (Wientjes & Nanninga 1991
) methods, lysis started a short time (10 and 20 min, respectively) after addition of the ß-lactam with no apparent connection to the division event. The key fact might be that the thymine/amino acid starvation synchronization method blocks division via FtsZ, whereas both the Baby Cell Machine and centrifugal elutriation methods do not. Therefore, in the former case no cell in the culture was able to trigger SMS before the end of the ongoing round of DNA replication. Addition of cefsulodin during the replication period surely affected diffuse lateral wall synthesis. However, as shown above, cells take a long time to lyse when only lateral wall synthesis is affected. Therefore, cells could terminate replication, trigger SMS and, as cefsulodin was present, a fast lysis would then follow. In Baby Cell Machine and centrifugal elutriation synchronized cultures alignment of division initiation is not quite as strict as it is for thymine/amino acid starvation where the cells have to go through a complete round of replication (around 40 min under the conditions used) before SMS can actually start. Furthermore, the doubling time in the experiments was relatively short (3060 min) and cefsulodin, as for any other ß-lactam, requires a few minutes before the onset of lysis. Therefore, it is quite possible that SMS was triggered in some cells before or shortly after addition of cefsulodin to the synchronized cultures even at the earlier times of drug addition. Then lysis could start soon after cefsulodin addition in all instances, as was indeed observed. The longer lysis delay observed in centrifugal elutriation synchronized cultures could reflect the better synchrony apparently obtained by this method.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 24 May 2001;
revised 20 August 2001;
accepted 15 September 2001.