Institut de génétique et de biologie microbiennes, Rue César-Roux 19, CH-1005 Lausanne, Switzerland1
Author for correspondence: Harold M. Pooley. Tel: +41 21 3206075. Fax: +41 21 3206078. e-mail: Harold.Pooley{at}igbm.unil.ch
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ABSTRACT |
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Keywords: poly(glycerol phosphate), tunicamycin, teichoic acid, thermosensitive tag mutants, phospholipid
Abbreviations: GlcNAc, N-acetylglucosamine; [2-3H]gro, [2-3H]glycerol; LTA, lipoteichoic acid; ND; nephelometric density; PG, peptidoglycan; P-lipid, phospholipid; poly(GlcGalNAc 1-P), poly(3-O-ß-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate); poly(groP), poly(glycerol phosphate); Ts, temperature sensitive; TUN, tunicamycin; WTA, wall teichoic acid
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INTRODUCTION |
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While most of these genes are known to be concerned with the synthesis of cell-surface components, knowledge of the interdependences of syntheses of the distinct surface components is limited to that of WTA and covalently linked PG (Mauck & Glaser, 1972 ). Notably, there is an almost total ignorance of the impact of interference with WTA on the synthesis of other cell-surface components, such as lipoteichoic acid (LTA) or phospholipid (P-lipid). It has been shown that blocking P-lipid synthesis in E. coli leads to a block in PG as well as of lipopolysaccharide synthesis (Ehlert & Höltje, 1996
). To adequately address these questions, a reliable method for the measurement of the kinetics of synthesis of relevant surface components in growing cells is needed; none has been reported so far. One such method is described here. Its use, in conjunction with some of the aforementioned mutants, has answered some of the questions raised above.
The exploitation of this method has provided a clearer picture of the effect on B. subtilis of tunicamycin (TUN), an antibiotic known to affect the synthesis of both PG and WTA (Ward et al., 1980 ). In vitro studies (Ward et al., 1980
) with membrane preparations revealed that, in the initial binding reaction of soluble precursor to lipid carrier, there was a fivefold greater sensitivity to TUN of the WTA-specific step as compared to the PG-specific one. However, data were inadequate to demonstrate that at the MIC, WTA was indeed the effective target in vivo. The cell fractionation method reported here for [2-3H]glycerol ([2-3H]gro)-labelled cell populations has been used to address the question of the in vivo target of TUN at the MIC for growth.
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METHODS |
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Growth conditions.
The protocol was devised for the study of Ts mutants, the phenotype of which is generally well expressed when exponentially growing cells are shifted to the non-permissive temperature. The increase was effected progressively over 1013 min to attenuate the heat shock (Pooley et al., 1991 ). Growth was expressed as nephelometric density (ND), measured with a Corning EEL nephelometer. An ND of 100 corresponds to approximately 108 cells ml-1 and a bacterial dry weight of 1·34 µg ml-1. Appropriately supplemented SAtrpgro medium was inoculated from a 13-d-old L plate and brought into exponential phase by overnight incubation at room temperature. This seed culture was diluted with the same medium to an ND of 4, incubated at 30 °C until ND 40, then progressively shifted to 47 °C and further incubated.
Radioactive labelling of cell surface components.
LTA, P-lipids and poly(groP), the major WTA (Sargent, 1973 ; Fischer et al., 1983
; Koch et al., 1984
), were labelled during growth at 30 and 47 °C in SAtrpgro (6 mM glycerol) supplemented with 1·5 µCi (5·55x104 Bq) ml-1 of [2-3H]gro. Samples (1 ml, generally), removed at regular intervals, were filtered, and the cells washed four times with prewarmed SAtrpgro containing 20 mM glycerol, and processed (see below).
To label the PG as well as poly(3-O-ß-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate) [poly(GlcGalNAc 1-P)], the minor WTA, cultures were grown in SAtrpgro medium supplemented with 100 µM GlcNAc and 0·2 µCi (7·4x103 Bq) ml-1 of [1-14C]GlcNAc. To follow incorporation into PG, samples, withdrawn at regular intervals, were filtered, and cells were washed with SAtrpgro containing 100 µM GlcNAc before being processed (see Results). Radioactive GlcNAc incorporation into poly(GlcGalNAc 1-P) was determined on separate samples according to Pavlik & Rogers (1973) as follows. Cell samples (25 ml) were collected on a membrane filter, washed well with water, and resuspended in 1 ml potassium phosphate buffer (0·1 M, pH 7·0). The soluble hexosamine nucleotide pool was extracted by a 5 min incubation in a boiling water bath. Cells were spun down and resuspended in 2 ml 0·1 M sodium citrate buffer pH 4·0. Extraction of poly(GlcGalNAc 1-P) was achieved by a 30 min incubation in a boiling water bath, and the soluble radioactivity separated by centrifugation from the bulk of the sedimentable radioactivity, corresponding to PG (Pavlik & Rogers, 1973
).
Fractionation of [2-3H]gro-labelled cells.
[2-3H]Gro-labelled cells, harvested on membrane filters and washed, were resuspended, by vigorous mixing, in 500 µl of TMS, the hypertonic buffer, containing 60 µg lysozyme. To ensure maximal solubilization of the cell-wall fraction, the suspension was generally incubated for up to 90 min at 37 °C. Total radioactivity was determined on a 50 µl aliquot. Protoplasts were sedimented at 15000 g for 15 min, and the radioactivity of the supernatant, containing the solubilized cell-wall fraction, was determined on a 400 µl aliquot, to which 1 ml water and 9 ml of a scintillation cocktail (Optifluor, Packard) were added. Following the removal of the supernatant, the protoplast pellet was washed by resuspension in 500 µl TMS, and resedimented. To extract the other, membrane-associated glycerol-containing cell components, i.e. LTA and P-lipid, the pellet, after being resuspended in 25 µl TMS with a toothpick, was mixed with 600 µl 0·2 M acetate buffer, pH 3·8, and 600 µl 80% aqueous phenol, and incubated with agitation for 60 min at 50 °C (modified from Fischer et al., 1983 ; Koch et al., 1984
). After cooling on ice, the aqueous and the phenol layers, containing LTA and P-lipid, respectively, were separated by a brief centrifugation, and the radioactivity present in each phase was measured.
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RESULTS |
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Consequences of incomplete cell-wall digestion
To determine adequate conditions for lysozyme digestion, [2-3H]gro- or [1-14C]GlcNAc-labelled exponentially growing cells were collected by filtration, washed, resuspended in the TMS lysis buffer and incubated at 37 °C. Samples, removed at regular time intervals, were centrifuged for 15 min at 15000 g and the radioactivity of the protoplast-free supernatant measured. After 45 min in the presence of lysozyme, inspection by phase-contrast microscopy revealed that over 95% of cells of the parent strain had been converted into protoplasts, whereas up to 90 min of lysozyme treatment was required for the maximum solubilization of the cell-wall-associated label, i.e. its complete release to the supernatant fraction (Fig. 1). Digestion for even longer periods was required in the case of certain tag (Ts) mutants after their transfer to 47 °C, the non-permissive temperature, and for cell walls made in the presence of TUN. The solubilization of the cell-wall fraction of [2-3H]gro-labelled cells followed a similar time course (data not presented) to that of [1-14C]GlcNAc-labelled cells (Fig. 1a
, b
).
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Selective extraction of LTA and of P-lipid
Following cell-wall digestion, the extraction procedure for P-lipid and LTA from [2-3H]gro-labelled protoplasts, on which we finally settled (see Methods), was chosen after examining two additional methods of processing the washed protoplast pellet. Initially, (i) the BlighDyer methanol/chloroform extraction of P-lipids from intact protoplasts (Rogers & Taylor, 1978 ), followed by biphasic aqueous phenol treatment to remove LTA, was used (Table 2
), and subsequently, (ii) an osmotic-shock-mediated lysis of protoplasts followed by the two-phase aqueous phenol extraction of LTA and P-lipids from cytoplasmic membranes obtained by centrifugation (Table 2
). Compared with the procedure finally adopted, both these methods were cumbersome, involved more manipulations and led to less accurate, more disperse measures. Nonetheless, they gave results substantially in agreement with those obtained with the simpler method finally adopted.
Synthesis of WTA, LTA and P-lipid in B. subtilis 168 and in mutants deficient in poly(groP) synthesis
To assess the reliability of the [2-3H]gro-labelling method, we determined the pattern of WTA synthesis, while simultaneously examining the kinetics of LTA and P-lipid synthesis, in several tag (Ts)-bearing mutants affected in one of the following enzymes: TagD, the glycerol-3-phosphate cytidylyltransferase; TagF, the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase; TagB, the putative CDP-glycerol N-acetylmannosamine glycerophosphotransferase, the enzyme adding two or more glycerol units from CDP-glycerol to the N-acetylmannosamine residue of the PG-WTA linkage unit (Mauël et al., 1994 ); or YvyH, the protein encoded by the previously characterized gene orfX (Soldo et al., 1993
). All but the last named were previously shown, after 2·5 h incubation at the restrictive temperature, to be deficient in WTA synthesis (Briehl et al., 1989
).
Following the protocol devised, exponentially growing cells of the 168 wild-type strain and Ts mutants were continuously labelled with [2-3H]gro and shifted to 47 °C. Samples, withdrawn at different times, were processed and the radioactivity corresponding to WTA, LTA and P-lipid fractions was measured. It appeared that during growth at 30 °C [2-3H]gro incorporation into all fractions paralleled overall mass increase (Fig. 2a). Following the temperature shift of the strain bearing the tag+ allele, the rate of incorporation into all fractions increased, as did the growth rate, although the relative incorporation into P-lipids and LTA was somewhat slower. At 47 °C, the tagF1-bearing strain was characterized by a rapid slow-down of [2-3H]gro incorporation into the WTA, an observation in full agreement with in vitro assays revealing an extremely Ts poly(groP) polymerase activity (Pooley et al., 1992
) exhibited by this mutant. However, incorporation into P-lipid and LTA continued upon the shift (Fig. 2b
), reaching, 20 min later, a rate parallel to that of mass increase. The two strains carrying mutated tagB (Fig. 2c
) or tagD (Fig. 2d
) alleles yielded results very comparable to those obtained with the tagF1-bearing strain (Fig. 2b
). Transfer to 47 °C provoked an immediate and strong reduction of the rate of isotope incorporation into poly(groP), whereas incorporation into P-lipid- and LTA-containing fractions continued, albeit at a slightly reduced rate relative to that of the wild-type strain.
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Results obtained with three further strains, bearing mutations in yvyH1 (orfX1) (Soldo et al., 1993 ), or tagF14 or rodB1, a mutation affecting a gene highly homologous to mreD of E. coli (Levin et al., 1992
; Varley & Stewart, 1992
), were for the last two barely distinguishable from those of the strain with the tag+ allele during 50 min of incorporation at the restrictive temperature (not presented). For the mutant affected in yvyH1, a somewhat reduced rate of incorporation for all glycerol-containing fractions was relatively more pronounced for the WTA during the initial 20 min at 47 °C (Fig. 2e
).
That the [2-3H]gro incorporation phenotype of the rodB1-bearing mutant was barely distinguishable from that of the strain with the tag+ allele is not surprising in that no WTA enzyme deficiencies were reported, neither was there any significant reduction in the phosphate content of cell walls prepared even after 2·53 h at 47 °C, i.e. after three to four times longer growth at the restrictive temperature than in the present incorporation studies. In strains with mutant yvyH1 and tagF14 alleles after 3 h at 47 °C, cell-wall phosphate was reduced but by only 10 and 40% (not presented), respectively, much less than the greater than 90% reduction observed in the case of the foregoing mutant alleles, tagB1, tagD1 and tagF1 (Briehl et al., 1989 ).
PG synthesis in strains bearing tag mutations
The method, as devised, providing relatively accurate kinetics of WTA synthesis, was exploited to examine the interdependence of WTA and PG synthesis. Cultures of the wild-type and of strains bearing mutant alleles of tagB, tagD or tagF growing exponentially in SAtrpgro medium supplemented with GlcNAc, were continuously labelled with [1-14C]GlcNAc. Aliquots collected at regular time intervals were split into two parts and treated as follows: (i) one series of samples was subjected to lysozyme digestion in hypertonic buffer, and the radioactivity in the solubilized cell wall (principally present in PG, but including that in the minor galactosamine-containing WTA), from which protoplasts were separated by centrifugation, was measured, and (ii) the other series was treated with a sodium acetate buffer (pH 4) to extract the poly(GlcGalNAc 1-P), the minor WTA of strain 168 (Estrela et al., 1991 ), as described in Methods. Incorporation kinetics (not presented) confirmed previous observations (Pooley et al., 1993
), i.e. the overall PG synthesis proceeds at rates comparable to that of the strain with the tag+ allele despite the inhibition of WTA synthesis.
Tunicamycin and cell wall synthesis
Finally, we investigated the effect of TUN on PG as well as on WTA synthesis in exponentially growing cultures of B. subtilis 168. Addition of 0·5 µg TUN ml-1 to a culture at an ND of 60, continuously labelled with [2-3H]GlcNAc at 30 °C, brought the synthesis of the minor WTA (Freymond, 1995 ) to a halt nearly instantaneously. Although the ND increase was slightly slowed down by TUN addition (Fig. 3
), [2-3H]GlcNAc incorporation into PG did not significantly differ from that in the control culture for at least 60 min, indicating that, under the experimental conditions used, the overall rate of PG synthesis was not affected. A similar experiment (Fig. 4
) with [2-3H]gro-labelled cells revealed, as expected, that the presence of TUN did not interfere for at least 80 min with the incorporation of 3H into the P-lipid and into LTA, which paralleled the ND increase. However, TUN addition provoked a marked reduction of poly(groP) synthesis. This finding was confirmed by a further experiment (not presented), in which inhibition of glycerol incorporation into poly(groP) became apparently complete 10 min after TUN addition. Examining cells by light microscopy 2 h after the drug addition revealed morphological changes resembling those characteristic of Ts WTA-deficient mutants, as reported previously (Takatsuki et al., 1972
). In conclusion, in confirmation of previous results obtained in in vitro studies, it would appear that in vivo TUN inhibits specifically the very first step of the WTA linkage unit systems, i.e. the coupling of GlcNAc to the lipid carrier. As had been predicted (Pooley & Karamata, 1994
), this is a reaction common to both the minor and the major WTA (Freymond, 1995
).
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DISCUSSION |
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The method described here for specifically measuring LTA synthesis offers a means of screening candidate mutants for those possibly deficient in the synthesis of this component. The current absence of LTA mutants, as well as of identified genes known to be involved in LTA synthesis, is a major handicap to the understanding of the biological role(s) of LTA, and of the regulation of its synthesis in relation to that of other surface components.
Analysis of [2-3H]gro incorporation by a strain bearing a Ts mutation in a distinct gene, rodB, not belonging to the tag gene cluster, whose expression provokes the development, under restrictive conditions, of morphological deformations comparable to those of tag-bearing mutants, revealed no significant reduction either in poly(groP) synthesis, or in that of the other glycerol-containing envelope components. This clear difference in response between mutations in tag genes and those deficient in rodB, a homologue of mreD of E. coli, associated with similar conditional-lethal morphological phenotypes, confirms that the so-called rod cell morphology developed by certain B. subtilis mutants may, but need not, be the result of inhibiting synthesis of the major WTA.
The rapid shut-off of WTA synthesis in tagF1-, tagB1- and tagD1-bearing mutants could be sufficient to cause the block in cell elongation. However, in the case of mutants such as tagF14 or yvyH1, inhibition of WTA is apparently neither rapid enough nor sufficiently pronounced to be considered as the primary cause of the defect which leads to the development of deformed swollen cells. Paradoxically, the kinetics of appearance of such deformed cells in the latter mutants are not significantly slower than in the mutants severely affected in WTA synthesis, revealing an absence of proportionality between the degree of inhibition of WTA synthesis and the extent of cell deformations.
The results can be reconciled, however, should the proteins concerned be constituents of a holoenzyme that includes enzymes involved in the synthesis of both major cell wall components, i.e. WTA and PG such that mutations affecting individual proteins could interfere with the normal working of the cell-wall elongation machinery, affecting proteinprotein interaction within the holoenzyme, without impeding, to a great extent, the activity of the catalytic site.
Like its E. coli homologue, the rodA gene of B. subtilis encodes a membrane protein that plays an essential role in the control of PG synthesis, ensuring elongation of the cylindrical part of the cells surface (Henriques et al., 1998 ). Such a protein could be an integral part of the elongation holoenzyme, although it has been suggested (Henriques et al., 1998
) that growth arrest could result from an inability of the swollen cells formed to initiate septation normally. However, the data provided, showing a shortening of the cells, in agreement with previous observations obtained on a tagF mutant (Pooley et al., 1993
), are consistent with a continued formation of septa, in contrast to the diminished rate of cell elongation, i.e. of the incorporation of cylindrical wall material, observed at the restrictive temperature. Since blocking cell elongation would provoke the greatest limitation of cell volume increase, we believe that the increased cell width that accompanies diminished activity of the rodA, rodB and tag genes reflects the tendency for the mass of cell cytoplasmic constituents to increase faster than the volume of rod-shaped cells.
In E. coli notably, the development of swollen cells can result from the temperature-dependent loss of activity of mutant protein(s) encoded by one of several genes, such as glmS, glmM and glmU, involved in GlcNAc biosynthesis, an intermediate common to both lipopolysaccharide and PG (Mengin-Lecreulx & van Heijenoort, 1996 ). Homologues of these genes are present in B. subtilis, where GlcNAc is an intermediate shared by WTA and PG. Although no B. subtilis mutants deficient in these genes have been examined, it will be interesting to know whether they are characterized by a conditional comparably swollen cell phenotype. Interestingly, mutants conditionally blocked downstream of GlcNAc formation, i.e. exclusively concerned in the synthesis of PG precursors, do not develop swollen cells. In B. subtilis (Brandt & Karamata, 1987
) as well as in E. coli (Mengin-Lecreulx & van Heijenoort, 1996
), they are all associated with cell lysis.
Experiments involving TUN at or near the MIC for growth of B. subtilis yielded a particularly clear and striking result in view of previous reports (Ward et al., 1980 ). During the 50 min following TUN addition, overall mass increase, the incorporation of labelled precursors into P-lipids and LTA and, most interestingly, into PG showed virtually no change, whereas incorporation into both the minor and the major WTA was rapidly and strongly reduced. This result (i) supports the suggestion (Pooley & Karamata, 1988
) that, at the MIC, WTA could be the growth-inhibitory target for TUN, and (ii) is in good agreement with results obtained in vitro with membrane preparations. Moreover, at a fivefold higher antibiotic concentration, the simultaneous inhibition of both WTA polymers (see above) and of PG (Ward et al., 1980
) is consistent with the UDP-GlcNAc translocase activity being the more sensitive target, since this enzyme is responsible for the first step in the biosynthesis of the linkage unit to PG.
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ACKNOWLEDGEMENTS |
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Received 15 October 1999;
revised 10 January 2000;
accepted 13 January 2000.