Eur. J. Biochem. 68, 271 - 280 (1976)

Monomer Addition as a Mechanism of Forming Peptide Cross-Links in the Cell-Wall Peptidoglycan of Streptococcus faecalis ATCC 9790 Eben H. OLDMIXON, Philippe DEZELEE, Marvin C. ZISKIN, and Gerald D. SHOCKMAN The Department of' Microbiology and Immunology and the Department of Radiology, Temple University School of Medicine, Philadelphia, Pennsylvania (Received May 4,1976)

The relative amounts of radioactively labeled disaccharide-peptide monomers and peptidecross-linked dimers and trimers found in the peptidoglycan of Streptococcus jaecalis ATCC 9790 were compared to the relative amounts to be expected from two different polymerization mechanisms (random condensation and monomer addition). Data from continuously-labeled, exponentially-growing cells are consistent with a monomer addition cross-linking process, not with a random condensation cross-linking mechanism. This conclusion was supported by data obtained from analyses of cells labeled during valine starvation (and wall thickening), recovery from valine starvation, and pulse and pulse-chase labeling of walls from exponentially-growing cultures

The native cell wall peptidoglycan is a network polymer which seems to be almost universally crucial in maintaining bacterial shape [l]. The basic repeating unit of that found in Streptococcusfueculis ATCC 9790 [I] is illustrated in Fig.1. A characteristic feature, with only a few exceptions, is a linear, unbranched P-1,4-linked polysacchdride composed of alternately repeating units of N-acetylglucosamine and N-acetylmuramic acid with peptide sidechains. Each disaccharide-peptide subunit is tetrafunctional and capable of bonding to as many as four other subunits. The hydroxyl on C-4 of N-acetylglucosamine and the hydroxyl on the C-I of N-acetylmuramic acid can each make P-1,4-glycosidic linkages with another subunit. The other two binding groups are located in the peptide sidechains. Chains of two subunits may be cross-linked by a peptide bind between the free carboxyl group of the penultimate D-alanine of one sidechain and a free a-amino group of the D-isoasparaginyl residue of another sidechain ; the terminal D-alanine residue (Fig. 2) iis released during the crosslinking process. Glycan chain polymerization proceeds by addition of activated subunits to pre-existing chains [2]. Biochemical investigations, however, have not yet clearly defined the process by which the peptide chains of peptidoglycans are cross-linked into dimers, trimers, tetramers, and higher oligomers.

H HC-OH

H

H

t H 0-7 H cH H

0 H

HC H

C=O

Hf *P H(D)C-C,

I

NH

HfH H

~tI H

c=o I

Fig. 1 . Chemicul structure of the disucc.hu,-ir~~~-~~~~i~ic/c. subunit o/ ilw type I I peptidoglyan in the classifi'cution of'Ghuysen ( I ] . such us is fbund in Streptococcus faecalis A T C C 9790. The subunit shown is N"-~-l,4-N-acetylglucosaminyl-N-acetylmuramyl-(~-alanyl-~-is~ glutaminyl)-N~-(~-isoasparaginyl)-~-lysl-~-alanyl-~-alanine. The u-isoasparaginyl residue serves as the cross-bridging amino acid between peptide sidechains. The N-acetylmuramyl residue is shown with 0-acetylation at the &carbon. (L) and (D) signify the L and D optical configurations about the anomeric carbons, respectively

212

Peptidoglycan Cross-Linking Mechanism

THEORY Random Cross-Linking : Mechanism and Resulting Distribution

The enzyme(s) which cross-link peptide chains could impinge upon the glycan scaffolding at random and form cross-links between suitably situated peptide sidechains. If all cross-linking events were independent from each other, then the peptides involved in a particular cross-linking event could be monomers, dimers, or longer peptide chains. This process, in which cross-linking would occur between any two chains, may be called ‘random condensation’ : Chain of x units

+ chain of y units Chain of (x + ,y) units

--f

where x,p = 1,2,3, . . . If all chains are equally reactive, regardless of length, then the weight fraction, W,, of chains of n subunits is [3] :

w, = npn-1 (1

- p)’

(1)

where W, = the weight fraction present as chains of n subunits, n = the number of subunits in a chain, p = the fraction of potential cross-links actually formed. The average chain length is :

x = l/(l

-

p)

(2)

where X = the average number of subunits in a chain, taken over all of the chains. The resulting distribution is commonly called the ‘most probable distribution’, because so many condensation schemes ultimately yield this final distribution [4].

An Alternative Mechanism : Monomer Addition

In contrast, monomers only might be added to acceptor groups on peptides of any length. Flory [ 5 ] demonstrated the salient properties of the ‘monomer addition’ mechanism: Chain of s units

+ monomer +

Chain of (x

+ 1) units

where x = 1,2,3, . . . The weight fraction of chains of length n would be given by :

where v = the average number of monomers added to the initiating units, considered over all of the chains. v need not be an integer. The average chain length, X, is [ 5 ] : X = v + l .

(4)

The abundances of peptide monomers, dimers, and trimers differ in S. .faecalis ATCC 9790 cell walls formed under differing conditions. This paper will show that peptide weight fraction distributions geneated by a monomer addition model resemble observed label distributions, while distributions resulting from the random condensation model do not; and we will show, also, that such a mechanism is consistent with our current knowledge of cell wall peptidoglycan synthesis. MATERIALS AND METHODS Cell growth conditions and the complete procedure for isolation, separation, identification, and quantitisation of [3H]lysine-labeled and [I4C]acetatelabeled peptidoglycan monomers and peptide-linked dimers, trimers, and oligomers have been described [6]. Random Condensation Mechanism : Predicted Monomer, Dinzer, and Trimer Weight Fractions

Flory [3] discussed the distributions associated with random condensation. Eqn (1) above) yields expressions for monomer, dimer, and trimer weight fractions : Monomer weight fraction: W I = (1 - p)’

( 5)

Dimer weight fraction: WZ = (1

-

p)’ (2p)

Trimer weight fraction: W3 = (1

-

p)’ ( 3 p 2 ) , (7)

(6)

Random Condensation Mechanism : Fitting Predicted Weight Fractions to the Observed Label Fractions in Monomer, Dimer, and Trimer Size Classes

In the above equations, a given value forp produces a unique series of values for the three weight fractions. We have used the xz (chi-squared) statistics to measure the goodness-of-fit between observed label fractions and values resulting from a particular p . For each data set, a single minimum x2 value exists (0 < p < 1). The equation that determines x2 as a function ofp is:

x2 = { [ W i(obs) - (1 - P ) ~ ] ’ / (1 - PI2 + [W2 (obs) - (1 - P)2(2P)12/ (1 - d 2 2 P + [W3 ( o w - (1 - P)2(3P2)12/ (8) (1 - p)’3p2}(1O0). The factor of 100 scales x2 for use with most statistical tables (see, for example [7]). Monomer Addition : Predicted Weight Fractions

The distributions associated with monomer addition have been developed by Flory [ 5 ] . Expressions

213

E. H. Oldmixon, P. Dezklee, M. C . Ziskin, and G. D. Shockman

for monomer, dimer, and trimer weight fractions are taken from Eqn (3) (above) : Monomer weight fraction : W1 = e-"/(v

+ 1)

Dimer weight fraction : w2 =@v) e-"/(v '

(9)

+ 1))

Trimer weight fraction : W3 ={(3v2/2)e-"/(v

+ 1)) .

(10) (11)

Monomer Addition Mechanism : Fitting Predicted Weight Fractions to the Observed Weight Fractions in the Monomer, Dimer, and Trimer Size Classes In this series of equations, the choice of a value for v produces a unique series of values for the three weight fractions. x2 was used to quantitise the degree of fit between observed fractions and those predicted by monomer addition. As noted previously, the appropriate single minimum in x2 exists. The following equation determines x2 as a function of v:

where

+ 1) Wz (exp) = v e-"/(v + 1) w3(exp) = v2 ep"/(2v + 2). W I (exp)

=

e-"/(v

Significance of Dissimilarities between Observed Label Fractions and Predicted Weight Fractions fir Random Condensation arzd Monomer Addition Mechanisms For both models, three weight fractions (monomer, dimer, trimer) were used to determine x 2 ; thus, the expected frequencies were estimated from the experimental observations. For such a case, the number of degrees of freedom is the number of categories reduced by two [8], giving one degree of freedom. The number of degrees of freedom is indicated in a subscript, c.g. xtl,. Tables comparing computed x2 values with the critical values in the x2 distribution may be found in a number of sources: Owen's Handbook of Statistical Tables [7] was used here, with graphical interpolation.

RESULTS Peptide Label Fraction Distributions in Peptidoglycan ,from Cells of S. faecalis Labeled Continuously during E.\cponential Growth The label fractions of monomers, dimers, and trimers from the peptidoglycan of continuously-labeled,

exponentially-growing cells are listed in Table 1, expt 1 A. The best fitting monomer addition and random condensation distributions (those with minimum x2) are compared graphically to the actual data in Fig. 2; only the monomer addition distribution models fit the observed distribution well. Table 1 also lists x2 values for these data and the corresponding probabilities that such values would occur by chance error if the respective models were in fact generating the data. The data from continuously-labeled, exponential phase cells differ significantly from the bestfitting random condensation distribution but not from the closest-fitting monomer addition distribution. The analysis of these experiments, in which exponentially-growing cells are labeled long enough to show the distribution of peptide chain lengths throughout the peptidoglycan, establishes the monomer addition model most firmly. Pep t ide Label Frac t ion D is tr ibu t ions in Peptidoglycanjrom Cells of's. faecalis Lubeled Continuously during Vdine Starvation

The peptidoglycan label fractions in continuouslylabeled, valine-starved cells and the fitting procedure results are listed in Table 1, expt 2 A . Here, too, the fit to a monomer addition model is substantially superior to a random condensdtion model. Peptide Label Fraction Distributions in Peptidoglycun j w m Cells o j S. faecalis Labeled by Pulses of'Increasing Length during Exponential Grmvth Table 1, expt B shows the data for these experiments and the results of analysis. As before, the monomer addition mechanism explains the data better than does the random condensation model. The xz value for the random condensation-observed data comparison does fall above the 0.05 level of significance, but the differences between the data and the best-fitting random condensation distribution become more significant as the pulses become longer. On the other hand, the monomer addition mechanism fits well at all pulse lengths. The mean peptide chain length increases with increasing pulse length (Fig. 3 A ) and reaches the level found in exponentially-growing walls after about 4 min. Peptide Label Fraction Distributions in Peptidoglycan @om Cells o f S. faecalis Labeled by a I-min Pulse and Chased.for Period.s o j Incrmsing Lengths In this series of experiments (Table 2), the fate of the older label is not masked by the introduction of newer, less cross-linked peptidoglycan. Random condensation cannot account for the observations, but

214

Peptidoglycan Cross-Linking Mechanism

Table 1. Label fractions of peptide monomers, dimers and (rimers isolated from S. faecalis peptidoglycan [6] and results of fitting random conclensution and monomer addition models to these duta The results of three types of experiments are listed. In expt 1 A, cells were labeled with [3H]lysine and [14C]acetatethrough eight generations of exponential growth. The experimental procedures, the technique of wall analysis, and results have been described (see [ 6 ] ,particularly Table 6). In expt 2A, cells were grown to an A h 7 5 of 0.8 (adjusted), collected, resuspended in medium lacking valine but containing [3H]lysine and [I4C]acetate, and maintained at 37 ‘C for 2 h. At the end of this period, the cells were harvested and their walls prepared and analyzed, In expts B, cells were labeled for increasing periods of time during exponential growth. The durations of the labeling periods used are indicated. The estimated degree of cross-linking (column hcadcd ‘Estimated p’) was calculated by the following equation: Estimated p = W ~ ( ~ h \ )f! 2 2 W3(obe)/3 f 4 1 1 - Wi t u b \ ) - WZ(obaJ - W 3 ( o b s ) } 5. The mean chain length associated with this degree of cross-linking is calculated with Eqn (2) (column headed ‘Estimated 2 nionomers, dinzers and tritners in pep tidoglycan front a continuouslj. labeled, c.uponcvtiully growing d t u r e . The actual data, presented both for the [3H]lysine and the [14C]acetate labels, are given by the left-most bar in each grouping of three. The central bars show the fraction given by the best-fitting (minimum value of I * ) monomer addition distribution, and the right-most bars show the bestfitting random condensation distribution

of cross-linking sufficient to maintain a relatively constant mean peptide chain length throughout the rest of the recovery period.

DISCUSSION Results presented in Table I show that monomer, dimer, and trimer weight fractions in the exponential phase and the valine-starved samples are significantly different from distributions predicted by a random condensation model. If the random condensation mechanism were in fact the dominant process in peptidoglycan cross-linking, the observed distributions would be expected to occur by chance fluctuations in fewer than one out of 100 trials (critical value = 0.005). In contrast, neithcr the exponential phase nor the valine-starved results differ significantly from

0

10

20

30

40

50

60

TIME AT START OF 4-MINUTE PULSE (minutes) A

3H

rn

'4c

Fig. 3. The ri~~ation.ship herxwn the rni'an chain length i (1,s d(,lernritwd h y the monnnw addition fittingpiocedurc,J und ( A ) durution of'pu1.w. ( B ) duration of' chases fdlowing a I-min pul.se of'lahrl, hotlr during eyionenrial growth, and ( C ) fi)r 4-min pulses during rec'ovrry f i o m vulinr .starvation. These data correspond to those in column 8, Table 1 (A), column 8 of Table 4 (B), and column X of Table 4 (C). The graphs shown in ( A ) and (B) indicate that the peptidoglycan is susceptible to additional crosslinking for a considerable length or time after its initial incorporation into the wall. (C) Indicates that during recovery from valine starvation an intitial stage in which the amount of cross-linking that may be accomplished in ii 4-min labeling period increases with increasing time into recovery; after this period there is a leveling-off stage in which the amount ofcrosslinking completed during 4-min pulses remains relatively constant

distributions expected from a monomer addition process. The lowest critical value that the exponentially-growing sample exceeds is 0.58; that for the valine-starved sample is 0.44. Clearly, the random condensation model does not explain the observed data satisfactorily. It is interesting that most schemes u here the formation, rearrangement, or dissolution o f inter-subunit bonds is at some time equiprobable (or almost so) though the population will yield distributions which are the same as or very similar to the ones resulting from random condensation [4], and consequcntly, such schemes are also improbable.

216

Peptidoglycan Cross-Linking Mechanism

Table 2. Data obtained when cells of S. faecalis ATCC 9790, growing erponentiall.v, ure pulsed w i f h 1ahel.for I rnin and t k m chased for various 1ength.r of time, as de,vrihiJdby DcClCr and Slioc,htnan ( 6 1 , and the results of the distribution7f2'ttin~procedure All details are described in the legend to Table 1 Duration Label of chase following 1-min pulse

Estimated

Estimated

P

.Ye

Random condensation model ~

s,

Monomer addition model ~~

~~

~

Xtl!

~

~~~

Pr

I ,

xi1!

Pr

min I4C

0.48 0.47

1.9 1.9

1.9 1.9

7.3 5.4

0.007 0.02

1.9 1.9

0.18 0.43

0.70 0.60

1

3H I4C

0.53 0.53

2.1 2.1

2.0 2.0

10 6.3

0.001 0.013

2.1 2.0

0.35 1.3

0.61 0.26

2

3H 14C

0.55 0.52

2.2 2.1

2.0 2.0

14 8.8

0.001 0.004

2.1 2.0

0.19 0.28

0.68 0.61

iH I4C

0.55 0.54

2.2 2.2

2.1 2.1

12 7.8

0.001 0.006

2.2 2.1

0.009 0.48

0.85 0.50

3H I4C

0.58 0.58

2.4 2.4

2.1 2.2

13 14

0.001 0.001

2.3 2.3

0.41 0.02

0.54 0.90

jH

0.59 0.59

2.4 2.4

2.2 2.2

15 17

0.001 0.001

2.4 2.3

0.16 0.25

0.65 0.65

0

4 8

10

3H

14C

Strictly speaking, one cannot assert that the monomer addition model is the correct explanation for the observed peptide label fraction distributions in these peptidoglycans. However, a best-fit test, such as applied here, can indicate relative likelihoods (or unlikelihoods) that observed data are consistent (or inconsistent) with various models. The observations d o not differ significantly from those expected by chance variation from monomer addition distributions, and this provides an acceptable, conceptually well-defined cross-linking mechanism. Although the mean peptide chain length in S. faecalis walls may seem low (usually about 2.0 subunits per chain), about one-fourth of the label is in chains with four or more units, and an appreciable number of longer chains exists. However, some influence must limit the cross-linking. Perhaps potentially reactive material is removed from regions of the growing cell with appropriate, active crosslinking enzymes by the production of new peptidoglycan in the vicinity of the ingrowing cross wall and movement of newly synthesized wall away from this region [9]. An analogous process has been proposed [lo] to explain the regulation of chain length during glycosaminoglycan biosynthesis. Chemical modifications might make subunits or chains unacceptable for cross-linking or inhibit the enzymes and thereby limit the active lifetimes of the components. Carboxypeptidase activities [l] or insertion of accessory wall polymers could serve such a function. Whatever regulates the lengths o f the peptide chains, the observed results are consistent with mono-

mer addition distributions. The results from exponentially-growing cells, however, are apparently in better agreement than the valine-starved cell results. This is evident in Table 1, where the minimum x2 values are listed and where the [14C]acetate data show the difference best. In those experiments where peptidoglycan from exponentially-growing cells was labeled by pulses of increasing length, the agreement between the observed distribution and a random condensation distribution worsens as the duration of the pulse increases (see Table 1). The minimum x2 value increases substantially, and the probability of chance perturbations in a random condensation distribution causing such results drops steadily to a very low level, suggesting a definite trend. Values from still longer pulses probably would have matched continuously labeled, exponentially-growing cell levels. Pulse-chase experiments (Table 2) allow one to follow the original label without interference from superimposed, new label. The results show that label incorporated during a relatively short pulse continues to show increasing mean peptide chain lengths for at least 32 min. Since these samples should have little or no new, sparsely cross-linked material, one would expect the mean chain length to be longer than in experiments without a chase period. This happens, and the mean chain lengths seen in the later samples are the highest reported in any of these experiments. The results of the analysis clearly favor monomer addition over random condensation in all samples; as expected, the fit provided by the random condensa-

E. H. Oldmixon, P. Deztlke, M. C. Ziskin, and G. D. Shockman

277

Table 3. Dciitr ohtaiizrtl when ~ 1 1 of s S. faecalis ATCC Y7Y0, starwd,fiw w l i n e utid allowed to r.ec'oivr, are ptrl.secl/iw pcriodr of 4 niiri ui 5-miii intervals during recorerq mid harvested immediaie1.v Also shown are the results of !he distribution-fitting procedure. The experiments are from Series A. All details are described in the legend to Table I

Time at Label star! of 4-min pulse

kstiinated P

Estimated

Raiidom condensation model -

I,

.I,

z1:

1

-

Monomer addition model -.

.

Pr

\-I,,

~

~

~

_

%ii J

Pr

min 0

3H I4C

0.45 0.45

1.8 1.8

1.8 1.8

2.6 3.1

0.11 0.08

I .7 1.7

0.96 0.79

0.34 0.38

5

H I4C

0.47 0.48

I.Y 1 .Y

1.9 1.9

5.3 5.1

0.03 0.03

1.8 1.9

0.40 0.51

0.53 0.48

'H 1 4 c

0.49 0.49

1 .Y 2.0

1.9 1.9

7.7 7.8

0.01. 0.01

I.8 1.9

0.9 1 0.63

0.34 0.43

'H I4C

0.48 0.49

1.9 2.0

1.9 1.9

5.8 5.0

0.02 0.03

1.9 1.9

0.42 0.83

0.52 0.38

3H

0.50 0.51

2.0 2.0

1.9

0.005 0.005

2.0 2.0

0.68 0.71

0.41 0.40

0.50 0.50

2.0 2.0

1.9 2.0

7.3 6.7

0.01 0.01

1.9 1.9

0.24 0.38

0.63 0.54

I4C

0.51 0.52

2.0 2.1

2.0 2.0

8.3 8.9

0.005 0.005

2.0 2.1

0.54 0.50

0.46 0.51

3H 14C

0.51 0.51

2.0 2.0

2.0 2.0

8.1

0.01

8.3

0.01

2.0 2.0

0.19 0.20

0.27 0.66

0.51 0.52

2.0 2.1

2.0 2.0

9.0

0.005 0.005

2.0 2.0

0.13 0.14

0.73 0.72

JH

0.50 0.52

2.0 2.1

1 .Y 2.0

0.005 0.005

2.0 2.0

0.19 0.28

0.67 0.61

3H

0.49 0.49

2.0 2.0

1.9 1.9

8.4 8.5

0.005 0.005

1.9 1.9

0.13 0.12

0.73 0.74

0.49 0.50

2.0 2.0

1.9 2.0

7.2 6.9

0.01 0.01

1.9 1.9

0.25 0.30

0.64 0.60

0.50 0.50

2.0 2.0

2.0 2.0

7.2 6.9

0.01 0.01

1.9 1.9

0.29 0.36

0.61 0.58

0.46 0.46

1.8 1.9

1.8 1.9

4.0 4.3

0.05 0.05

1.8 1.8

0.58 0.78

0.45 0.38

10 15

20

14C

25

3H I4C

'H

30 35 40

3H 1

45

50

4

14~7

55

'H 14c

60

3H I4c

65

3H 14C

~

I .9

tion mechanism deteriorates with increasing chase time, while the monomer addition mechanism remains consistent with the observations. The agreement between observed and monomer addition distributions may improve somewhat over the 1-10-min range of pulse lengths shown (see the data pertaining to the 14C label given in Table l), but the x2 values indicate that the data do not deviate significantly from monomer addition distributions. So, while 6-min, 8-min or 10-min pulses might show the conditions of the total peptide chain population more faithfully, 4-min pulses (Table 3) are sufficiently long to show the same resemblance to monomer addition distributions which is observed after extended labeling periods.

14 15

9.4 13 14

Frere et al. [ l l ] have established that, in the Streptomyces R61 system, only the activated, ~ - A l a D-Ala end of the peptide sidechain on a monomer serves as the donor group and that the N-terminal end of the peptide sidechains on either monomers or growing chains serve as acceptors. In the StveptococC U S system, it remains unclear whether a monomeric peptide may be added to either end of a pre-existing chain. If the terminal D-alanine has been cleaved by carboxypeptidase activity, leaving only a single, carboxy-terminal D-alanine, then addition by the typical cellular enzymes probably will not occur at that end. However, where addition to either end remains chemically possible, one does not yet know whether some device, presumably relying on the discriminatory

278

Peptidoglycaii Cross-Linking Mechanism

Table 4. Data obtained when cells of' S. f'aecalis ATC'C' 9790, starved fbr valine and allowe~Ito recover, (Ire pul.wri,fbr periods of'4 min a t 5-min intervals during recovery and hurvested imrnediately Also shown are the results of the distribution-fitting procedure. The experiments are from Series B. The data shown in this table was obtained in a second series of experiments which used the same procedure uscd to obtain the data shown in Table 3. All details are described in the legend to Table 1 Timeat start of 4-mm pulse

Label

F\tiinnted I)

Estimdtcd

Rdndoin condeiisdtion model ~

~~

~~

Monomer addition model ._

~

\L

yr

/?I

I

~-

~

~

Pr

ym

Y?l 1

Pr

~

min 0

0.39 0.40

1.7 1.7

1.7 1.7

5.5 3.7

0.02 0.06

1.6 1.7

0.07 0.32

0 79 0 59

5

0.42 0.44

1.7 1.8

I .7 1.8

3.6 4.6

0.06 0.08

1.7 1.7

0.33 0.32

0 57 0 58

10

0.44 0.45

1.8 1.8

1.8 1.8

2.2 2.6

0.14 0.11

1.7 1.7

1.1 1.1

0 29 0 31

15

0.45 0 45

1 .s 1.8

1.8 1.8

6.2 4.8

0.02 0.03

1.8 1.8

0.15 0.41

0 71 0 53

20

0.48 0.47

1.9 1.9

1.9 1.9

6.1 5.2

0.02 0.03

1.8 1.8

0.47 0.57

0 50 0 45

25

0.47 0.46

1.9 1 .s

1.9 1.x

5.2 5.3

0.03 0.04

1.8 1.8

0.44 0.37

0 51 0 55

30

0.48 0.48

1.9 1.9

1.9 1.8

4.2 3.2

0.05 0.03

1.8 1.8

1.0

1.4

0 33 0 23

35

0.47 0.47

1.Y 1.9

1.9 1.8

5.8 7.0

0.02 0.07

1.8 1.8

0.30 0.27

0 61 0 61

40

0.50 0.49

2.0 2.0

1.8 1.9

4.5 4.6

0.04 0.04

1 .8 1.8

0.70 0.69

0 40 0 41

0.47 0.47

1.9 1.Y

1.9 1.9

9.1 9.1

0.005 0.005

1.8 1.9

0.16 0.21

0 70 0 66

45

0.49 0.50

2.0 2.0

1.9 1.9

7.0 6.9

0.01 0.01

1.9 1 .Y

0.75 0.79

0 19 0 38

50

0.51 0.50

2.0 2.0

2.0 2.0

13 12

0.002 0.005

2.0 2.0

0.10 0.02

0 75 0 88

55

0.49 0.50

1.9 2.0

1 .9

9.0 10

0.005 0.005

1.9 1.9

0.40 0.18

0 53 0 67

0.51 0.52

2.0 2.1

1.9 1 .Y

17

0.001 0.001

2.0 2.0

0.55 1.0

0 46 0 32

0.50 0.50

2.0 2.0

I .9 1.9

0.001 0.003

1.9 1.9

0.67 0.46

0 42 0 52

60 65

1.9

capacities of an enzyine(s), directs addition to a particular end. The weight fraction distribution would be the same whether addition were allowed at only one end or at both ends, so the distribution data do not exclude either possibility. Obviously, monomer addition itself is not new: many biological polymerizations involve chain elongation through addition of individual. monomeric subunits. The biopolymerizations of ribonucleic acid. or the amylose chains in glycogen and starch, for example, proceed by a tailward elongation mechanism. that is, one in which the energy-carrying part of a subunit condenses with the tail end of a growing

19

8.2 9.6

chain; this newest subunit becomes, in turn, the acceptor for the next addition. Fatty acids, terpenes, proteins [12], and the glycan chains of peptidoglycan [2] are synthesized by a monomer addition mechanism also, but the growth direction is reversed. Lipmann states: 'An activated terminal is kept in front of the growing chain as the result of a particular arrangement ; the activated chain head reacts with the tail of a single unit only if this already carries the activating annex on its head' [12]. In at least one other biological system, chain length distribution data have been used to evaluate informally the type of polymerization mechanism ; this has been done with

E. H. Oldmixon, P. Dezelee, M. C. Ziskin, and G. I). Shockman

chondroitin sulphates, and the distributions there, too, have appeared to be closer to monomer addition distributions [lo]. However, the observation that a sheet-like polymer, such as peptidoglycan, can be formed by two chemically different, monomer addition polymerizations proceeding in different directions seems particularly interesting and favors particular peptidoglycan assembly models. The experiments of Mirelman et al. [13] demonstrated that Micrococcus luteus peptidoglycan precursor material was first gelled into an insoluble product when peptide cross-links were formed. The Bacillus licheniJbrrni.7 system of Ward and Perkins [2] showed that the polymerization of soluble glycan strands could occur under some conditions independently of their incorporation by cross-linking into an insoluble product. Only one cross-link would be required to incorporate a linear chain into a preexisting, insoluble network, and only two cross-links per chain, on the average, would be required to form a true, branching network [4]. If the length of the glycan strands in peptidoglycan is estimated (conservatively) at 75 - 100 disaccharide-peptide subunits in length [14], then about 1 cross-linking should suffice to make an insoluble product, and 2 to 3 to form a gelled network. Cross-linking, it seems, occurs to an extent far greater than that which would simply be necessary for strand incorporation. The additional tightening of the network would improve structural properties (strength, rigidity, and elasticity) and therefore is probably not superfluous. Our results show that the bulk of peptidoglycan cross-linking, in excess of that required for glycan strand gelation, apparently occurs by monomer addition. Whether the small amount of cross-linking actually necessary to make glycan strands insoluble proceeds by another mechanism cannot be determined here, but it seems unlikely, and in fact, our analyses show no division of crosslinking into gelling (early) and strengthening (late) mechanisms. These observations have been combined into a sketch of the possible relationships between peptidoglycan synthetic processes in Fig. 4, which should be compared with Fig.5 of Mirelman et nl. [13]. As suggested by Ward and Perkins [2], the elongation of the glycan strands, requiring a reducing end with attached undecaprenol pyrophosphate, is pictured as membrane-associated. That our use of the basic monomer addition and random condensation equations in the context of growing peptidoglycan is valid is seen in the following informal arguments. It does not matter that the subunits are not free to mix. At any stage of the reaction, the frequency of occurrence of monomers. dimers, trinicrs, (it(.., as. say, right-hand neighbors of monomers will reflect

219 KEY TO SUBUNIT STRUCTURE END; C-4 OF N-ACETYL G L UCOSAMINE DISACCHARIDE PEPTIDE SIDE CHAIN (N-TERMINAL TO C-TERMINAL)

\REDUCING END; C-1 OF N-ACETYLMURAMIC ACID UNDECAPRENOL PYROPHOSPHATE

MEMBRANE

Fig. 4. Shcicli qf /iy/ioihi~ticu/I u j w of'/,c,pii[lo,slycrrll t l r u w .SO (1.5 to he c~orr.~i.~ic~nt n,it/r S. I'aecalis auponrwtiullj. , ~ r o i i ~ i i i ,c'e q cr.os.~-lid~ing, fi.cyucvwir.\ o/ peptirlc, nionon find longer. o/igoi?zrr.s) nnd w i t h rstinicitc~.rof Icngt/i. The older section is on the riglit: on the left, disaccharidepeptide subunits are being polymerized into glycan chains in ii meiiibrane-associated process. The chains are being fixed into the peptidoglycan net by transpeptidation (a monomer addilion process) in the middle. Sinall arrows indicate sites where additional bonds might be formed. The directions of strand alignment and approach are quite arbitrary. since the true spatial relationship between the disaccharide and the peptide within a subunit is not yet known. (1) Indicates addition of subunit to reducing end of xccptor strand. (2) release of undecaprenol pyrophocpliate moiety and ( 3 ) crosslinking reaction (transpeptidation). proceeding by inonoiner addition

their overall frequency of occurrence in the population, which is the important condition. As the cross-linking polymerization proceeds, the overall rate of reaction in any sinall segment of the growing wall may be expected to slow, because of the decreased number of monomers, chemical niodification of the substrate, the decreased number of active enzymes, or like causes. However, if one imagines two containers of monomers at different temperatures, both polymerizing with a monomer

280

E. H. Oldmixon, P. Dezelee, M. C. Ziskin, and G. D. Shockman: Peptidoglycan Cross-Linking Mechanism

addition mechanism, one sees that they would react at different rates, but if the reactivities of the various species generated are all equally (or nearly so) affected by the temperature difference, then a monomer addition (modified Poisson) distribution would result in each container. Therefore, monomer addition distributions would result at intermediate temperatures, too. Similarly, if the temperature of one container were changed during the reaction, the rate of the overall reaction would change, but the distribution type would be preserved. This situation is analogous to that at the growth zones of the cell, and the overall frequency distribution of species formed reflects the underlying mechanism of polymerization. Similar arguments are equally applicable to the random condensation mechanism. The authors would like to acknowledge gratefully the expert technical assistance of Mr John C. Mazza, who performed the experiments in Series B, the pulse-chase experiments and others. They also wish to thank D r P. Flory for his instructive comments on the manuscript. This work was supported by United States Public Health research grant AI-05044. E.H.O. was a recipient of a Predoctoral Fellowship (GM-49468) from the National Institutes of Health.

REFERENCES 1. Ghuysen, J.-M. & Shockman, G . D. (1973) in Bacterial Memhranes and Wa1l.s (Leive, L., ed.) pp. 37- 130, Dekker, New York. 2. Ward, J. B. & Perkins, H. R. (1973) Biockem. J . 135,721 -728.

3. Flory, P. J . (1936) J . Am. Chem. SOC.58, 1877-1885. 4. Flory, P. J. (1953) Principles of Polymer Chemistry, Cornell University Press, Ithaca, New York. 5. Flory, P. J. (1940) J . A m . Chem. Soc. 62, 1561 -1565. 6. Dezelee, P. & Shockman, G . D . (1975) J. Biol. Chen7. 250, 6806-6816. 7. Owen, D. B. (1962) Handbook of Statistical Tables. AddisonWesley Publ. Co., Reading, Massachusetts. 8. Colquhoun, D. (1971) Lectures on Bios/a/ixtics,Oxford Univers-

ity Press, London. 9. Higgins, M . L. & Shockman, G. D. (1970) J . Bacteriol. 101, 643 - 648. 10. Hopwood, J. J. & Robinson, H. C. (1973) Biochem. J . 135, 631 -637. 11. Frere, J.-M., Ghuysen, J.-M., Zeiger, A. R. & Perkins, H. R. (1976)FEBSLert. 63, 112-116. 12. Lippmann, F. (1968) Essays Biochrm. 4 , 1-23. 13. Mirelman, D., Bracha, R. & Sharon, N. (1972) Proc. Nut1 Acad. Sci. U . S . A .69, 3355-3359. 14. Rogers, H . J. (1974) Ann. N . Y . Acud. Sci. 235, 29-51.

E. H. Oldmixon, Lehrstuhl Mikrobiologie 11, lnstitut fur Biologie 11. Eberhard-Karls-Universitat Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Federal Republic of Germany P. DeztlCe, Institut de Radiobiologie, Universite de Paris-Sud, F-91405 Orsay, France M . C. Ziskin, Department of Radiology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania, U.S.A. 19140 G. D . Shockman, Department of Microbiology and Immunology, Temple University School of Medicine, 3420 North Broad Street, Philadclphia, Pennsylvania, U.S.A. 19140