Archives of

Microbiology

Arch. Microbiol. 121, 177-180 (1979)

9 by Springer-Verlag 1979

Covalent Linkage of Lipoprotein to Peptidoglycan is not Essential for Outer Membrane Stability in Proteus mirabilis Jobst Gmeiner Institut f/Jr Mikrobiologie, Fachbereich Biologie, Technische Hochschule Darmstadt, Schnittspahnstr. 9, D-6100 Darmstadt, Federal Republic of Germany

Abstract. Isolated rigid layers from Proteus mirabil& harvested at different growth phases were degraded by endo-N-acetylmuramidase fi'om Chalaropsis B, and the degradation products were investigated. The results show the complete absence of covalently linked lipoprotein in exponential-phase cultures. Stationary cells, however, possess covalently linked lipoprotein in a m o u n t s similar to those f o u n d in Escheriehia coli or Salmonella typhimurium during all growth phases. The overall peptidoglycan structure did not change during transition f r o m logarithmic to stationary growth. Implications o f these findings for the organization of the outer m e m b r a n e are discussed.

Key words: Proteus mirabilis - Covalently linked lipoprotein -

G r o w t h phase -

Outer membrane.

,Cell walls o f m o s t gram-negative enteric bacteria contain a rigid layer in which a specific lipoprotein is linked covalently to the structural polymer peptidoglycan (murein) (for review see Braun, 1975), However, Proteus mirabilis harvested in the logarithmic growth phase was reported to lack such a lipoprotein (Braun et al., 1970). Rigid layers isolated f r o m stationary cells o f this organism, on the other hand, showed a granular appearance in the electron microscope, similar to rigid layers isolated f r o m Escherichia coll. These granules or particles disappeared after trypsin treatment, and, therefore, were regarded as the covalently linked protein (Martin, ]964) which was later identified as lipoprotein. Recently, covalently linked lipoprotein was isolated f r o m rigid layers of Proteus mirabilis grown to early stationary phase, and was shown to be very similar chemically to the E. coli lipoprotein (Gmeiner et al., 1978). However, only a b o u t every 80th peptidoglycan

subunit was substituted with one lipoprotein molecule in contrast to E. coli, where lipoprotein was f o u n d at about every 10th peptidoglycan subunit (Braun, 1975). In the present c o m m u n i c a t i o n it will be shown that covalently linked lipoprotein in P. mirabilis is indeed present only in stationary cells, confirming fully the previous findings cited above (Braun et al., 1970; Martin, 1964). The peptidoglycan structure, on the other hand, and the degree o f crosslinking remains the same independent o f lipoprotein attachment.

Materials and Methods Bacterial Strain and Culture Conditions. Proteus mirabilis strain 19, obtained from Prof. Martin (this laboratory), was cultivated in aerated liquid complex beef extract-tryptic casein peptone medium as described previously (Martin et al., 1975). Cells from the logarithmic phase were harvested after 2.5 h at A578 = 0.85 ; stationary phase cells were harvested after 16 h at A578 > 4. Cells grown to early stationary phase in a fermenter were purchased from E. Merck (Darmstadt, Germany). Rigid Layer Isolation. Cell walls were prepared by shaking bacterial suspensions in 0.4 % sodium dodecylsulfate solution (w/v) with glass beads. Rigid layers were usually isolated from the cell walls after repeated extraction with 4 % sodium dodecylsulfate (w/v) at 100~C as previously described (Gruss et al., 1975; Braun and Rehn, 1969). It is of interest to note that rigid layers from log cells containing no covalently linked lipoprotein (see Results) were much more difficult to 'sediment by ultracentrifugation from the sodium dodecylsulfate solution than lipoprotein containing rigid layers. For the isolation of rigid layers from stationary phase cultures cell walls were first extracted with 0.01 M Tris/HC1 buffer, pH 7.5, containing 0.001 M EDTA, 1.5 % sodium dodecylsutfate (w/v) and 0.01% sodium azide (w/v) at 100~C before treatment with 4 % sodium dodecylsulfate. The rigid layer preparations were washed twice with 0.1 M NaC1, dialyzed extensively against distilled water and stored in the frozen state. Enzymic Degradation of Rigid Layers and Separation of the Fragments. Rigid layers were degraded with endo-N-acetylmuramidase from Chalaropsis B, kindly provided by Dr. J.H. Hash (Vanderbilt University), and the degradation products were separated by gel filtration in 0.1 M LiC1on Sephadex G50 and G25 according to Fleck et al., 1971 as described in detail previously

0302-8933/79/0121/0177/$01.00

178

Arch. Microbiol., Vol. 121 (1979)

(Gmeiner et el., 1978). Quantitative determination of the peptidoglycan degradation products was performed by amino acid analysis after acid hydrolysis (Gmeiner et al., 1978).

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Rigid layers prepared from Proteus mirabilis cells harvested at different growth phases were degraded by endo-N-acetylmuramidase from ChalaropsisB, since this enzyme also cleaves glycosidic linkages of Nacetylmuramic acid substituted by O-acetyl groups peculiar to P. mirabilis peptidoglycan (Fleck et el., 1971; Martin and Gmeiner, 1979). The soluble degradation products were separated from insoluble material by low speed centrifugation and filtered over serially connected columns of G 50 and G 25 in 0.1 M LiC1 (Fig. 1). Rigid layer fragments containing lipoprotein were eluted with the void volume, well separated from the other degradation products, namely disaccharide peptide trimers, dimers and monomers. Fractions containing the various fragments were pooled and analyzed by amino acid analysis. The percentage distribution of meso-2,6-diaminopimelic acid in the various fragments as a measure for peptidoglycan subunits is given in Table 1. In the case of rigid layers obtained from exponential-phase cells only 0.2% of the total recovered meso-2,6-diaminopimelic acid eluted with the void volume and no UV absorbing material could be detected. In contrast, rigid layers obtained from stationary cells yielded more than 10 % peptidoglycan subunits in this fraction. This increase was not due to insufficient cleavage of the rigid layers by the enzyme but rather to increased amounts of fragments containing lipoprotein, since amino acid analysis of this fraction gave comparatively higher contents of amino acids found in lipoprotein. In addition, amino acids not present in P. mirabilis lipoprotein such as glycine, phenylalanine or histidine (Gmeiner et al., 1978) were found only in minor quantities and sodium dodecylsulfate polyacrylamide gel-electrophoresis showed no contamination with other proteins except Chalaropsis endo-N-acetylmuramidase. It should be noted that although P. mirabilis lipoprotein is quite soluble in water enzymatic degradation of rigid layers derived from stationary cells yielded some insoluble material of unknown composition (Gmeiner et al., 1978). Less than 1% of the total meso2,6-diaminopimelic acid was recovered with this insoluble material of three different preparations. Despite the difference in lipoprotein attachment the peptidoglycan structure of the various cell preparations did not change significantly. As evident from the molar ratio of disaccharide peptide monomers, crosslinked dimers and trimers, the degree ofcrosslinkage remained

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Fig. 1. Separation of rigid layer degradation products by gel filtration. 10ml of a solution containing rigid layer degradation products from stationary cells, equal to 1801]mol meso-2,6diaminopimelic acid, were applied to serially connected columns of Sephadex G 50 and Sephadex G 25 (gel bed volume 400 ml each) equilibrated in 0.1 M LiC1 (Fleck et al., 1971). The columns were eluted with 0.1 M LiC1 at a flow rate of 56ml/h. The effluent was monitored by a differential refractometer from Winopal (Hannover, Germany) and by an Uvicord from LKB (Bromma, Sweden). 5 ml fractions were collected.

Table 1. Percentage distribution of meso-2,6-diaminopimelic acid in rigid layer fragments recovered after separation by gel filtration (Fig. 1) Rigid layer preparation

Void Trimer volume fraction

Dimer fraction

Monomer fraction

% Logarithmic cells

0.2

8.0

49.2

42.6

Early stationary cells preparation 1 preparation 2

1.6 1.8

5.2 8.7

44.6 41.8

48.6 47.7

Stationary cells preparation 1 preparation 2 preparation 3

10.8 10.5 10.0

5.8 7.6 10.0

39.9 44.7 44.5

43.5 37.2 35.5

rather constant (Table2). Furthermore, thin layer chromatography of the isolated fragments from the various rigid layer preparations revealed a comparable degree of O-acetylation of N-acetylmuramic acid residues (not shown). The only difference observed was about 50% loss of the terminal D-alanine in the disaccharide peptide monomer fraction derived from stationary rigid layers. Peptidoglycan fragments remaining attached to the lipoprotein showed a similar ratio of monomers and dimers as determined previously (Gmeiner et al., 1978).

J. Gmeiner: Covalent Lipoprotein in Proteus mirabilis

179

Table2. Molar ratio of peptidoglycan fragments recovered after separation by gel filtration (Fig. 1) Rigid layer preparation

Monomers Dimers Trimers Mol/mol

Logarithmic cells Early stationary cells preparation 1 preparation 2 Stationary cells preparation 1 preparation 2 preparation 3

1.00

0.58

0.06

1.00 1.00

0.46 0.44

0.04 0.06

1.00 1.00 1.00

0.46 0.60 0.63

0.05 0.07 0.09

Discussion The results presented show clearly that Proteus mirabilis is capable of synthesizing lipoprotein and linking it covalently to peptidoglycan during the stationary growth phase only. This synthesizing capacity has also been demonstrated by Katz et al. (1978). These authors reported the puromycin resistance of lipoprotein biosynthesis in P. mirabilis as previously established for Escherichia coli, and succeeded in the isolation of free lipoprotein. Concerning free lipoprotein, it should be mentioned that sodium dodecylsulfate polyacrylamide gel-electrophoresis of cell wall proteins from the various growth phases showed no significant variation in the amount of free lipoprotein (Gmeiner, in prep.). Since covalently linked lipoprotein remains attached to disaccharide peptide monomers as well as to crosslinked dimers after enzymic rigid layer degradation, it was determined that only about 70 % of the meso-2,6-diaminopimelie acid recovered with the lipoprotein fraction represents lipoprotein attachement sites (Gmeiner et al., 1978). Hence, rigid layers from early stationary phase cells were calculated to contain lipoprotein molecules at about every 80th peptidoglycan subunit (Gmeiner et al., 1978). Similar calculations for rigid layers from stationary cells (Table 1) establish lipoprotein attachment at about every 14th peptidoglycan subunit. This figure is quite comparable to the amount oflipoprotein found with rigid layers ofE. coli (every 10th-- 12th peptidoglycan subunit; Braun, 1975) or Salmonella typhimurium (every 9 t h - 1 4 t h peptidoglycan subunit; Braun et al., 1970; Gmeiner and Schlecht, 1979) independent of the growth phase. The complete absence oflipoprotein attached to the peptidoglycan sacculus in exponential-phase cells of P. mirabilis therefore leads to the conclusion that covalently linked lipoprotein is not as essential for cell wall stability in this organism as it apparently is in E. coli. This may be an indication for an important

difference in outer membrane organization in the two enteric bacteria. In E. coli the outer membrane is believed to be anchored to the underlying peptidoglycan network by the covalently linked lipoprotein (Braun and Rehn, 1969). Furthermore, it was claimed that lipoprotein is indispensible for cell division in this organism (Torti and Park, 1976; Weigand et al., 1976), and Yamada and Mizushima achieved reconstitution of an ordered structure from outer membrane constituents only on lipoprotein-bearing peptidoglycan sacculi (Yamada and Mizushima, 1978). On the other hand, E. coli mutants either lacking lipoprotein completely (Hirota et al., 1977), or synthesizing modified lipoprotein (Yem and Wu, 1978) display functional deficiencies of their outer membrane. In P. mirabilis the O-acetylation of part of the muramic acid residues conferring higher hydrophobicity to the rigid layer probably suffices for the association of outer membrane components to the peptidoglycan layer in exponentially growing cells. In this respect it will be of interest to study the association between the outer membrane and the peptidoglycan in the penicillin-induced spheroplast L-form of P. mirabilis, where a considerable decrease of Oacetylation of muramic acid residues was observed in the peptidoglycan still synthesized during penicillin action (Martin and Gmeiner, 1979). Acknowledgements. The author is thankful to Prof. H. H. Martin for

his generous support and to Miss Hildegard Bergmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft.

References Braun, V.: Covalent iipoprotein from the outer membrane of Escherichia coli. Biochim.Biophys.Acta (Amst.) 415, 335 - 377 (1975) Braun, V., Rehn, K. : Chemicalcharacterization,spatial distribution and function of a lipoprotein (murein-lipoprotein)of the E. coli cell wall. Eur. J. Biochem. 10, 426-438 (1969) Braun, V., Rehn, K., Wolff,H. : Supramolecularstructureof the rigid layer of the cell wall of Salmonella, Serratia, Proteus, and Pseudomonasfluorescens. Number of lipoproteinmoleculesin a membrane layer. Biochemistry9, 5041-5049 (1970) Fleck, J., Mock, M., Minck, R., Ghuysen,J. M. : The cellenvelopein Proteus vulgaris P18. Isolation and characterization of the peptidoglycancomponent.Biochim.Biophys.Acta (Amst.)233, 489-503 (1971) Gmeiner, J., Kroll, H.-P., Martin, H. H. : The covalent rigid-layer lipoproteinin cellwalls of Proteus mirabilis. Eur. J. Biochem.83, 227-233 (1978) Gmeiner, J., Schlecht, S. : Molecular organization of the outer membrane of Salmonella typhimurium. Eur. J. Biochem. 93, 609-620 (1979) Gruss, P., Gmeiner, J., Martin, H. H. : Amino-acidcompositionof the covalent rigid-layer lipoprotein in cell walls of Proteus mirabilis. Eur. J. Biochem.57, 411-414 (1975)

180 Hirota, Y., Suzuki, H., Nishimura, Y., Yasuda, S. : On the process of cellular division in Escherichia coli - Mutant of E. coli lacking a murein-lipoprotein. Proc. Natl. Acad. Sci (Wash.) 74, 14171420 (1977) Katz, E., Loring, D., Inouye, S., Inouye, M.: Lipoprotein from Proteus mirabilis. J. Bacteriol. 134, 674-676 (1978) Martin, H. H. : Composition of the mucopolymer in cell wall~ of the unstable and stable 1_-form of Proteus mirabilis. J~ Gen. Microbiot. 36, 441-450 (1964) Martin, H. H., Maskos, C., Burger, R.: D-Alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis. Eur. J. Biochem. 55, 465-473 (1975) Martin, H. H., Gmeiner, J. : Modification of peptidoglycan structure by penicillin action in cell walls of Proteus' mirabilis. Eur. J. Biochem. in press 1979

Arch. Microbiol., Vol. 12/(1979) Torti, S. V., Park, J. T. : Lipoprotein of gram-negative bacteria is essential for growth and division. Nature (Lond.) 263, 323 - 326 (1976) Weigand, R. A., Vinci, K. D., Rothfield, L. I. : Morphogenesis of the bacterial division septum: A new class of septation-defective mutants. Proc. Natl. Acad. Sci. (Wash.) 73, 1882-1886 (1976) Yamada, H., Mizushima, S. : Reconstitution of an ordered structure flom major outer membrane constituents and the lipoproteinbea'ing peptidoglycan sacculus of Esckerichia coti. J. Bacteriol. 135, 1024-1031 (1978) Yem, D. W., Wu, H. C : Physiological characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein. J. Bacteriol. 133, 1419-1426 (1978) Received January 29, 1979

Covalent linkage of lipoprotein to peptidoglycan is not essential for outer membrane stability in Proteus mirabilis.

Archives of Microbiology Arch. Microbiol. 121, 177-180 (1979) 9 by Springer-Verlag 1979 Covalent Linkage of Lipoprotein to Peptidoglycan is not Es...
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